LOCUS R1_Bxb1_ZH2A_vas 6827 bp DNA circular SYN 13-MAY-2025 DEFINITION . ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM recombinant plasmid REFERENCE 1 (bases 1 to 6827) AUTHORS Junjie Luo TITLE Direct Submission JOURNAL Exported May 13, 2025 from SnapGene 8.0.3 https://www.snapgene.com FEATURES Location/Qualifiers source 1..6827 /mol_type="other DNA" /organism="recombinant plasmid" misc_feature 64..251 /label=2xr4_PCR /note="color: #a6acb3" misc_feature 64..80 /label=attPBxb1_r4_syn /note="color: #a6acb3" promoter join(81..96,101..116,121..136,141..156) /label=r4 promoter /note="This forward directional feature has 4 segments: 1: 81 .. 96 / #ffffff 2: 101 .. 116 / #ffffff 3: 121 .. 136 / #ffffff 4: 141 .. 156 / #ffffff" promoter join(163..178,183..198,203..218,223..238) /label=r4 promoter /note="This forward directional feature has 4 segments: 1: 163 .. 178 / #ffffff 2: 183 .. 198 / #ffffff 3: 203 .. 218 / #ffffff 4: 223 .. 238 / #ffffff" promoter join(279..284,285..290,318..403) /label=hsp70 mini /note="Drosophila melanogaster hsp70Bb promoter" /note="This forward directional feature has 3 segments: 1: 279 .. 284 / #ffffff 2: 285 .. 290 / #cc99ff / TATA box 3: 318 .. 403 / #ffffff / align to 3xP3" regulatory 417..426 /label=Kozak sequence /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" /note="color: #a6acb3" /regulatory_class="other" CDS join(423..425,426..428,429..1142) /codon_start=1 /product="enhanced GFP" /label=EGFP /note="mammalian codon-optimized" /note="This feature has 3 segments: 1: 423 .. 425 / #05fd14 2: 426 .. 428 / #05fd14 / 1a 3: 429 .. 1142 / #05fd14" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" polyA_signal 1283..1404 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" /note="color: #a6acb3" promoter 1447..1867 /label=dU6-3 promoter /note="RNA polymerase III promoter for Drosophila U6-3 snRNA (Port et al., 2014)" /note="color: #ffffff; direction: RIGHT" misc_feature 1870..1889 /label=attB_gRNA(core) /note="color: #00ff00; direction: RIGHT" misc_RNA 1890..1975 /label=LgRNA scaffold /note="long gRNA, removed tttt, extended loop guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" /note="color: #00ccff; direction: RIGHT" primer_bind complement(2004..2020) /label=SK primer /note="common sequencing primer, one of multiple similar variants" /note="color: #a020f0; direction: LEFT" misc_feature complement(2037..2324) /label=PhiC31 attB /note="color: #a6acb3; direction: LEFT" protein_bind complement(2239..2308) /label=attB /bound_moiety="phage phi-C31 integrase" /note="attB site for the phi-C31 integrase (Groth et al., 2000)" /note="color: #31849b; direction: LEFT" misc_feature complement(join(2310..2312,2313..2315,3973..3989)) /label=split attB_SgrDI_gRNA target /note="This reverse directional feature has 3 segments: 1: 2310 .. 2312 / #00ff00 2: 2313 .. 2315 / #00ff00 3: 3973 .. 3989 / #00ff00" misc_feature join(2316..2338,2339..3490,3491..3516,3517..3574,3575..3581, 3582..3967,3968..3971) /label=[AD1_P^{Bxb1}] /note="This forward directional feature has 7 segments: 1: 2316 .. 2338 / #ffffff 2: 2339 .. 3490 / #ff0000 / 2xr4-mCherry 3: 3491 .. 3516 / #ffffff 4: 3517 .. 3574 / #ff00ff / attP[Bxb1] 5: 3575 .. 3581 / #ffffff 6: 3582 .. 3967 / #31849b 7: 3968 .. 3971 / #ffffff" polyA_signal 2339..2460 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" /note="color: #a6acb3" CDS complement(2467..3177) /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /note="color: #ff0000" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" promoter complement(join(3188..3273,3301..3306,3307..3312)) /label=hsp70 mini /note="Drosophila melanogaster hsp70Bb promoter" /note="This reverse directional feature has 3 segments: 1: 3188 .. 3273 / #ffffff / align to 3xP3 2: 3301 .. 3306 / #cc99ff / TATA box 3: 3307 .. 3312 / #ffffff" misc_feature 3320..3507 /label=2xr4_PCR /note="color: #a6acb3" promoter complement(join(3333..3348,3353..3368,3373..3388, 3393..3408)) /label=r4 promoter /note="This reverse directional feature has 4 segments: 1: 3333 .. 3348 / #ffffff 2: 3353 .. 3368 / #ffffff 3: 3373 .. 3388 / #ffffff 4: 3393 .. 3408 / #ffffff" promoter complement(join(3415..3430,3435..3450,3455..3470, 3475..3490)) /label=r4 promoter /note="This reverse directional feature has 4 segments: 1: 3415 .. 3430 / #ffffff 2: 3435 .. 3450 / #ffffff 3: 3455 .. 3470 / #ffffff 4: 3475 .. 3490 / #ffffff" misc_feature complement(3517..3574) /label=attP(Bxb1) /note="http://flybase.org/reports/FBto0000358.html The attP(Bxb1) site (5'-GTCGTGGTTTGTCTGGTCAACCACCgcggtctcAGTGGTGTACGGTACAAACCCC GAC-3') is one of the two target sites of the Bxb1:int integrase. Bxb1:int mediates recombination between two non-identical target sites, attP(Bxb1) and attB(Bxb1), each of which contains a short integration core flanked by imperfect inverted repeats. Exchange between the attP(Bxb1)/attB(Bxb1) pair results in the formation of hybrid sites that are no longer a substrate of the Bxb1:int integrase, and thus the exchange reaction driven by Bxb1:int is undirectional (reviewed in PMID:21195181, FBrf0231034). The recombination event catalyzed by Bxb1:int results in genetic modification, the nature of which is influenced by the relative orientation (direct or inverted), and location of the two target sites. The types of possible modification include deletion of DNA, generation of chromosomal rearrangements, integration of DNA into the genome, and replacement of genetic material with that from a donor plasmid using recombination-mediated cassette exchange (RMCE) (reviewed in FBrf0231034)." /note="color: #ff00ff; direction: LEFT" protein_bind 3582..3967 /label=gypsy insulator /bound_moiety="Su(Hw), CP190, andMod(mdg4)67.2" /note="chromatin insulator from Drosophila" /note="color: #31849b" misc_feature 3973..5033 /label=PhiC31_vasa3_Arm /note="color: #a6acb3" misc_feature complement(join(3973..3986,3987..3988,3989)) /label=attB_gRNA (miss match) /note="The original attB gRNA is ACCGTCGACGATGTAGGTCANGG. The 2nd and 3rd nt are miss matched. " /note="This reverse directional feature has 3 segments: 1: 3973 .. 3986 / #00ff00 2: 3987 .. 3988 / #c0c0c0 / miss match 3: 3989 .. 3989 / #00ff00" misc_feature complement(3999..4573) /label=vasa 3' UTR /note="color: #953ce6; direction: LEFT" CDS complement(4592..4639) /codon_start=1 /product="bipartite nuclear localization signal from nucleoplasmin" /label=nucleoplasmin NLS /note="color: #993366" /translation="KRPAATKKAGQAKKKK" CDS complement(4640..5032) /codon_start=1 /product="integrase from phage phi-C31" /label=phage phi-C31 integrase partial sequence /note="color: #993366" /translation="YEDRAAGAYDGPVGRKHFRKQQAALTLRQQGAEERLAELEAAEAP KLPLDQWFPEDADADPTGPKSWWGRASVDDKRVFVGLFVDKIVVTKSTTGRGQGTPIEK RASITWAKPPTDDDEDDAQDGTEDVAA" rep_origin complement(5094..5682) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" /note="color: #ffff00" CDS complement(join(5853..6644,6645..6713)) /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /note="This feature has 2 segments: 1: 5853 .. 6644 / #ccffcc 2: 6645 .. 6713 / #ccffcc / signal sequence Cleavage site after base 6644" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(6714..6818) /gene="bla" /label=AmpR promoter /note="color: #ffffff; direction: LEFT" ORIGIN 1 ttaattaact cgcgttaaga tacattgatg agtttggaca aacctgcagg ccctttcgtc 61 ttcctaactt ccgtgacccg ttaaaataat caggcgtaga ttaaaataat caggcggtca 121 ttaaaataat caggcggaga ttaaaataat caggcgatgc atttaaaata atcaggcgta 181 gattaaaata atcaggcggt cattaaaata atcaggcgga gattaaaata atcaggcgag 241 cggagactct aagcttatcg atttcgaacc ctcgaccgcc ggagtataaa tagaggcgct 301 tcgtctacgg agcgacaatt caattcaaac aagcaaagtg aacacgtcgc taagcgaaag 361 ctaagcaaat aaacaagcgc agctgaacaa gctaaacaat cggctcgaag ccggtcgcca 421 ccatggtgag caagggcgag gagctgttca ccggggtggt gcccatcctg gtcgagctgg 481 acggcgacgt aaacggccac aagttcagcg tgtccggcga gggcgagggc gatgccacct 541 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca 601 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga 661 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct 721 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc 781 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc 841 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga 901 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg 961 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc 1021 actacctgag cacccagtcc gccctgagca aagaccccaa cgagaagcgc gatcacatgg 1081 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt 1141 aaagcggcct aagcgatcgc tcaggccgcg actctagatc ataatcagcc ataccacatt 1201 tgtagaggtt ttacttgctt taaaaaacct cccacacctc cccctgaacc tgaaacataa 1261 aatgaatgca attgttgttg ttaacttgtt tattgcagct tataatggtt acaaataaag 1321 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt 1381 gtccaaactc atcaatgtat cttacctgca ggagtaaact cgtcgacggt atcgataagc 1441 gctagctttt ttgctcacct gtgattgctc ctactcaaat acaaaaacat caaattttct 1501 gtcaataaag catatttatt tatatttatt ttacaggaaa gaattccttt taaagtgtat 1561 tttaacctat aatgaaaaac gattaaaaaa aatacataaa ataattcgaa aatttttgaa 1621 tagcccaggt tgataaaaat tcatttcata cgttttataa cttatgcccc taagtatttt 1681 ttgaccatag tgtttcaatt ctacattaat tttacagagt agaatgaaac gccacctact 1741 cagccaagag gcgaaaaggt tagctcgcca agcagagagg gcgccagtgc tcactacttt 1801 ttataattct caacttcttt ttccagactc agttcgtata tatagaccta ttttcaattt 1861 aacgtcggta ccgtcgacga tgtaggtcag tcttagagct atgctggaaa cagcatagca 1921 agttaagata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcttttt 1981 tatcgaattc ctgcagcccg ggggatccac tagttctaga gcggcgcgcc catatgttcg 2041 gcttgtcgac atgcccgccg tgaccgtcga gaacccgctg acgctgcccc gcgtatccgc 2101 acccgccgac gccgtcgcac gtcccgtgct caccgtgacc accgcgccca gcggtttcga 2161 gggcgagggc ttcccggtgc gccgcgcgtt cgccgggatc aactaccgcc acctcgaccc 2221 gttcatcatg atggaccaga tgggtgaggt ggagtacgcg cccggggagc ccaagggcac 2281 gccctggcac ccgcaccgcg gcttcgagac cgtgacctac atcgtctcga gacctaggta 2341 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa atgctttatt 2401 tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa taaacaagtt 2461 accggtttac ttgtacagct cgtccatgcc gccggtggag tggcggccct cggcgcgttc 2521 gtactgttcc acgatggtgt agtcctcgtt gtgggaggtg atgtccaact tgatgttgac 2581 gttgtaggcg ccgggcagct gcacgggctt cttggccttg taggtggtct tgacctcagc 2641 gtcgtagtgg ccgccgtcct tcagcttcag cctctgcttg atctcgccct tcagggcgcc 2701 gtcctcgggg tacatccgct cggaggaggc ctcccagccc atggttttct tctgcattac 2761 ggggccgtcg gaggggaagt tggtgccgcg cagcttcacc ttgtagatga actcgccgtc 2821 ctgcagggag gagtcctggg tcacggtcac cacgccgccg tcctcgaagt tcatcacgcg 2881 ctcccacttg aagccctcgg ggaaggacag cttcaagtag tcggggatgt cggcggggtg 2941 cttcacgtag gccttggagc cgtacatgaa ctgaggggac aggatgtccc aggcgaaggg 3001 cagggggcca cccttggtca ccttcagctt ggcggtctgg gtgccctcgt aggggcggcc 3061 ctcgccctcg ccctcgatct cgaactcgtg gccgttcacg gagccctcca tgtgcacctt 3121 gaagcgcatg aactccttga tgatggccat gttatcctcc tcgcccttgc tcaccatgtt 3181 gacgcgtccg attgtttagc ttgttcagct gcgcttgttt atttgcttag ctttcgctta 3241 gcgacgtgtt cactttgctt gtttgaattg aattgtcgct ccgtagacga agcgcctcta 3301 tttatactcc ggcggccgct agagtctccg ctcgcctgat tattttaatc tccgcctgat 3361 tattttaatg accgcctgat tattttaatc tacgcctgat tattttaaat gcatcgcctg 3421 attattttaa tctccgcctg attattttaa tgaccgcctg attattttaa tctacgcctg 3481 attattttaa cgggtcacgg aagttagcat gcaagcgtcg gggtttgtac cgtacaccac 3541 tgagaccgcg gtggttgacc agacaaacca cgacgagctc gaattgatcg gctaaatggt 3601 atggcaagaa aaggtatgca atataataat cttttattgg gtatgcaacg aaaatttgtt 3661 tcgtcaacgt atgcaatatt ctttattaaa agagggtatg caatgtattt tattaaaaac 3721 gggtatgcaa tataataatc ttttattggg tatgcaacga aaatttgttt cgtcaaagta 3781 tgcaatattt tttattaaaa gagggtatgc aatgtatttt attaaaaacg ggtatgcaat 3841 aaaaaattat ttggtttctc taaaaagtat gcagcactta ttttttgata aggtatgcaa 3901 caaaatttta ctttgccgaa aatatgcaat gtttttgcga ataaattcaa cgcacactta 3961 ttacgtgggc cccctacatc gtcgacacta gtggatccaa cacgaagagc agcagtgtgg 4021 tggatttctc agttattgat tttgcttctg caaagacagc aaacgcaaaa gggcaacatt 4081 tgctggtcgg catgcttttc gggggaggag gactgggcgc gagcagggcg aggcatgcat 4141 tcaaaaagcc ggctccaacg gagagcgaga gagcgcaaga gccaagtgta cagcaaggca 4201 aacaacaaac aacgaaacac gaaggatgag tacttaaaac caaacacgtg atagtacacg 4261 gtattagttg ggatcaaagt gcacgtttct gttaagtttt gcaaccgctc gttattaaaa 4321 gcagttcaca aaatacttaa aagaaatcga attagagcca gaacattttt tcaaattata 4381 ttttgttata catttctttg taaaaggaaa ttaaaataga aaattcgaat ggcttgcata 4441 tcctaaatat gcattttatt attatatgcg ttataataca tataattaat atgttattaa 4501 tatctaactt atttaactaa ttacatcaat tactgcatta catttaatta tttgaaatct 4561 atgtccatac attgagctca ggcctagatt actttttctt ttttgcctgg ccggcctttt 4621 tcgtggccgc cggccttttc gccgctacgt cttccgtgcc gtcctgggcg tcgtcttcgt 4681 cgtcgtcggt cggcggcttc gcccacgtga tcgaagcgcg cttctcgatg ggcgttccct 4741 ggcccctgcc cgtagtcgac ttcgtgacaa cgatcttgtc tacgaagagg ccgacgaaca 4801 cgcgcttgtc gtctacgctc gcgcggcccc accacgactt agggccggtc gggtcagcgt 4861 cggcgtcttc ggggaaccat tggtcaaggg gaagcttcgg ggcttcggcg gcttcaagtt 4921 cggcaagccg ctcttccgcg ccttgctgcc ggagcgtcag cgctgcctgt tgcttccgga 4981 agtgcttcct gccaacgggt ccgtcgtacg cgcctgccgc gcggtcttcg taacatgtga 5041 gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat 5101 aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac 5161 ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct 5221 gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg 5281 ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg 5341 ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt 5401 cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg 5461 attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac 5521 ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga 5581 aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt 5641 gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt 5701 tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga 5761 ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc 5821 taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct 5881 atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata 5941 actacgatac gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca 6001 cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga 6061 agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga 6121 gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg 6181 gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga 6241 gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt 6301 gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct 6361 cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca 6421 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat 6481 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga 6541 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc 6601 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg 6661 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc 6721 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt 6781 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttcc //