LOCUS R1_Bxb1_ZH2A_nos 6831 bp DNA circular SYN 13-MAY-2025 DEFINITION . ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM recombinant plasmid REFERENCE 1 (bases 1 to 6831) 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..6831 /mol_type="other DNA" /organism="recombinant plasmid" misc_feature 64..251 /label=2xr4_PCR /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" 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 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" 5'UTR complement(3999..5036) /label=nos-3'UTR /note="color: #a6acb3; direction: LEFT" rep_origin complement(5098..5686) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" /note="color: #ffff00" CDS complement(join(5857..6648,6649..6717)) /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: 5857 .. 6648 / #ccffcc 2: 6649 .. 6717 / #ccffcc / signal sequence Cleavage site after base 6648" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(6718..6822) /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 gtggatccca agtggcacaa tgtgggcgac 4021 aaggactatc ccaatattgg caagaacgtc ctgtattgcc agccggacag tgatcttacc 4081 gatggcatct tcctggccct tttcgagaaa cgccgcgagg gcgaaaagga ttagttgttt 4141 caaacgcaag aaggacattt gtttccttaa attgtaacca tttctttatt tggcactcga 4201 gccattgaat ttttcatttt cagaatatgt gtacacattt tttaaaaaaa taaaaaaatt 4261 atataatgct ggcggttgtt tcatgtgtga aaaattgatc aatggtaaac aaaattgaat 4321 aaatatataa catatatata tatatatata tatatatata tatatagata tgtgtgttga 4381 aatgaatact tgcgatacat gtaataaaaa tactcttcgc ttatctatca aaaagtgcgg 4441 aatgtcaaaa tttaaaattt tacaatgaat gcgtagccga cgacgaaagt gttccttgct 4501 atttccttta gcaagattta aatttagatt aaattctaat gatacgattg acagttcgaa 4561 attcaaagtg ttcctttttc aaaatttagt aaagattgta tatcaattgt agatatatcg 4621 aaatttttcg gccgcaagcg aacattttac aaaatgaagg cgaccagttg cagaccaatt 4681 ccattcatca actttcggat tgtaagatat ttctatcggc cacgacgatt gaacaagtat 4741 tacgatattg taagtcttct ttaacaaaat tagtttccct ttcacagaaa cagacataaa 4801 ttcttgaatt attgacttgg atttgagtga tcgttcgttg tctatactat aagatctata 4861 ggcacgggat aacgctctaa atctctttaa aaccgaacgc gccaggcgct agttaaacgt 4921 tactatctat ctatctggtt aacccagctt tgatcggaat gcgtatatat atttcatgtt 4981 atataaacgc tgcaaaagct gccagagcct ctgctccaga gctggattcg ccctctacat 5041 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt 5101 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg 5161 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc 5221 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt 5281 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa 5341 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta 5401 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa 5461 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa 5521 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt 5581 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt 5641 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat 5701 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat 5761 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc 5821 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc 5881 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta 5941 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga 6001 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg 6061 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc 6121 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat 6181 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag 6241 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat 6301 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa 6361 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa 6421 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga 6481 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg 6541 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc 6601 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg 6661 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact 6721 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat 6781 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc c //