LOCUS R2_Bxb1_ZH2A_pBS 6298 bp DNA circular SYN 13-MAY-2025 DEFINITION . ACCESSION . VERSION . KEYWORDS . SOURCE synthetic DNA construct ORGANISM recombinant plasmid REFERENCE 1 (bases 1 to 6298) 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..6298 /mol_type="other DNA" /organism="recombinant plasmid" rep_origin complement(51..479) /direction=LEFT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" /note="color: #ffff00" primer_bind 621..637 /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" /note="color: #a020f0; direction: RIGHT" promoter 644..662 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" /note="color: #ffffff; direction: RIGHT" misc_RNA complement(722..807) /label=LgRNA scaffold /note="guide RNA scaffold for the Streptococcus pyogenes CRISPR/Cas9 system" /note="color: #00ccff; direction: LEFT" misc_feature complement(join(808..810,811..827)) /label=pacman_R2_gRNA_15_0_off /note="This reverse directional feature has 2 segments: 1: 808 .. 810 / #00ff00 2: 811 .. 827 / #00ff00" promoter complement(829..1249) /label=dU6-3 promoter /note="RNA polymerase III promoter for Drosophila U6-3 snRNA (Port et al., 2014)" /note="color: #ffffff; direction: LEFT" polyA_signal 1292..1413 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" /note="color: #a6acb3" CDS complement(1534..2211) /codon_start=1 /product="rapidly maturing tetrameric variant of DsRed fluorescent protein (Bevis and Glick, 2002)" /label=DsRed-Express /note="mammalian codon-optimized" /note="color: #ff0000" /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK LKVTKGGPLPFAWDILSPQFQYGSKVYVKHPADIPDYKKLSFPEGFKWERVMNFEDGGV VTVTQDSSLQDGSFIYKVKFIGVNFPSDGPVMQKKTMGWEASTERLYPRDGVLKGEIHK ALKLKDGGHYLVEFKSIYMAKKPVQLPGYYYVDSKLDITSHNEDYTIVEQYERAEGRHH LFL" regulatory 2208..2217 /label=Kozak sequence /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" /note="color: #a6acb3" /regulatory_class="other" promoter complement(join(2231..2316,2344..2349,2350..2355)) /label=hsp70 mini /note="Drosophila melanogaster hsp70Bb promoter" /note="This reverse directional feature has 3 segments: 1: 2231 .. 2316 / #ffffff / align to 3xP3 2: 2344 .. 2349 / #cc99ff / TATA box 3: 2350 .. 2355 / #ffffff" misc_feature 2387..2574 /label=2xr4_PCR /note="color: #a6acb3" promoter complement(join(2400..2415,2420..2435,2440..2455, 2460..2475)) /label=r4 promoter /note="This reverse directional feature has 4 segments: 1: 2400 .. 2415 / #ffffff 2: 2420 .. 2435 / #ffffff 3: 2440 .. 2455 / #ffffff 4: 2460 .. 2475 / #ffffff" promoter complement(join(2482..2497,2502..2517,2522..2537, 2542..2557)) /label=r4 promoter /note="This reverse directional feature has 4 segments: 1: 2482 .. 2497 / #ffffff 2: 2502 .. 2517 / #ffffff 3: 2522 .. 2537 / #ffffff 4: 2542 .. 2557 / #ffffff" protein_bind 2580..2649 /label=attB /bound_moiety="phage phi-C31 integrase" /note="attB site for the phi-C31 integrase (Groth et al., 2000)" /note="color: #31849b; direction: RIGHT" misc_feature complement(join(2726..2728,2729..2731,3995..4011)) /label=split pacman_R2_gRNA_15 target /note="This reverse directional feature has 3 segments: 1: 2726 .. 2728 / #00ff00 2: 2729 .. 2731 / #00ff00 3: 3995 .. 4011 / #00ff00" misc_feature complement(join(2732..2759,2760..3919,3920..3925,3926..3975, 3976..3994)) /label=[AD2_B^{Bxb1}] /note="This reverse directional feature has 5 segments: 1: 2732 .. 2759 / #ffffff 2: 2760 .. 3919 / #00ffff / 2xr4-CFP 3: 3920 .. 3925 / #ffffff 4: 3926 .. 3975 / #31849b / attB[Bxb1] 5: 3976 .. 3994 / #ffffff" misc_feature 2743..2930 /label=2xr4_PCR /note="color: #a6acb3" promoter join(2760..2775,2780..2795,2800..2815,2820..2835) /label=r4 promoter /note="This forward directional feature has 4 segments: 1: 2760 .. 2775 / #ffffff 2: 2780 .. 2795 / #ffffff 3: 2800 .. 2815 / #ffffff 4: 2820 .. 2835 / #ffffff" promoter join(2842..2857,2862..2877,2882..2897,2902..2917) /label=r4 promoter /note="This forward directional feature has 4 segments: 1: 2842 .. 2857 / #ffffff 2: 2862 .. 2877 / #ffffff 3: 2882 .. 2897 / #ffffff 4: 2902 .. 2917 / #ffffff" promoter join(2937..2942,2943..2948,2976..3061) /label=hsp70 mini /note="Drosophila melanogaster hsp70Bb promoter" /note="This forward directional feature has 3 segments: 1: 2937 .. 2942 / #ffffff 2: 2943 .. 2948 / #cc99ff / TATA box 3: 2976 .. 3061 / #ffffff / align to 3xP3" CDS join(3072..3074,3075..3077,3078..3791) /codon_start=1 /product="cyan variant of GFP" /label=CFP /note="mammalian codon-optimized" /note="This feature has 3 segments: 1: 3072 .. 3074 / #00ffff 2: 3075 .. 3077 / #00ffff / 1a 3: 3078 .. 3791 / #00ffff" /translation="MVSKGEELFTGVVPILVELDGDVNGHRFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTWGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYISHNVYITADKQKNGIK AHFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" polyA_signal 3798..3919 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" /note="color: #a6acb3" misc_feature 3926..3975 /label=attB(Bxb1) /note="http://flybase.org/reports/FBto0000359 The attB(Bxb1) site (5'-TCGGCCGGCTTGTCGACGACGgcggtctcCGTCGTCAGGATCATCCGGGC-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: #31849b; direction: RIGHT" misc_feature 3976..3981 /label=SpeI /note="color: #a6acb3" misc_feature 4107..4112 /label=SpeI /note="color: #a6acb3" promoter complement(4131..4149) /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" /note="color: #ffffff; direction: LEFT" primer_bind complement(4170..4186) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" /note="color: #a020f0; direction: LEFT" protein_bind 4194..4210 /label=lac operator /bound_moiety="lac repressor encoded by lacI" /note="The lac repressor binds to the lac operator to inhibit transcription in E. coli. This inhibition can be relieved by adding lactose or isopropyl-beta-D-thiogalactopyranoside (IPTG)." /note="color: #31849b" promoter complement(join(4218..4224,4225..4242,4243..4248)) /label=lac promoter /note="promoter for the E. coli lac operon" /note="This reverse directional feature has 3 segments: 1: 4218 .. 4224 / #ffffff / -10 2: 4225 .. 4242 / #ffffff 3: 4243 .. 4248 / #ffffff / -35" protein_bind 4263..4284 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." /note="color: #31849b" rep_origin complement(4572..5160) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" /note="color: #ffff00" CDS complement(join(5331..6122,6123..6191)) /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: 5331 .. 6122 / #ccffcc 2: 6123 .. 6191 / #ccffcc / signal sequence Cleavage site after base 6122" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" promoter complement(6192..6296) /gene="bla" /label=AmpR promoter /note="color: #ffffff; direction: LEFT" ORIGIN 1 catttccccg aaaagtgcca cctaaattgt aagcgttaat attttgttaa aattcgcgtt 61 aaatttttgt taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta 121 taaatcaaaa gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc 181 actattaaag aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg 241 cccactacgt gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact 301 aaatcggaac cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt 361 ggcgagaaag gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc 421 ggtcacgctg cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc 481 ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg cctcttcgct 541 attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg taacgccagg 601 gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttgtaatacg actcactata 661 gggcgaattg gacatgttac cgtcgacgat gtaggccggg ctgcaggaat tcgataaaaa 721 agcaccgact cggtgccact ttttcaagtt gataacggac tagccttatc ttaacttgct 781 atgctgtttc cagcatagct ctaagaccga aaccgctggg cgcggtgccg acgttaaatt 841 gaaaataggt ctatatatac gaactgagtc tggaaaaaga agttgagaat tataaaaagt 901 agtgagcact ggcgccctct ctgcttggcg agctaacctt ttcgcctctt ggctgagtag 961 gtggcgtttc attctactct gtaaaattaa tgtagaattg aaacactatg gtcaaaaaat 1021 acttaggggc ataagttata aaacgtatga aatgaatttt tatcaacctg ggctattcaa 1081 aaattttcga attattttat gtattttttt taatcgtttt tcattatagg ttaaaataca 1141 ctttaaaagg aattctttcc tgtaaaataa atataaataa atatgcttta ttgacagaaa 1201 atttgatgtt tttgtatttg agtaggagca atcacaggtg agcaaaaaag ctagcgctta 1261 tcgataccgt cgacgagttt actcctgcag gtaagataca ttgatgagtt tggacaaacc 1321 acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc tattgcttta 1381 tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat tcattttatg 1441 tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt 1501 ggtatggctg attatgatct agagtcgcgg cccctacagg aacaggtggt ggcggccctc 1561 ggcgcgctcg tactgctcca cgatggtgta gtcctcgttg tgggaggtga tgtccagctt 1621 ggagtccacg tagtagtagc cgggcagctg cacgggcttc ttggccatgt agatggactt 1681 gaactccacc aggtagtggc cgccgtcctt cagcttcagg gccttgtgga tctcgccctt 1741 cagcacgccg tcgcgggggt acaggcgctc ggtggacgcc tcccagccca tagtcttctt 1801 ctgcattacg gggccgtcgg aggggaagtt cacgccgatg aacttcacct tgtagatgaa 1861 ggagccgtcc tggagggagg agtcctgggt cacggtcacc acgccgccgt cctcgaagtt 1921 catcacgcgc tcccacttga agccctcggg gaaggacagc ttcttgtagt cggggatgtc 1981 ggcggggtgc ttcacgtaca ccttggagcc gtactggaac tggggggaca ggatgtccca 2041 ggcgaagggc agggggccgc ccttggtcac cttcagcttg gcggtctggg tgccctcgta 2101 ggggcggccc tcgccctcgc cctcgatctc gaactcgtgg ccgttcacgg agccctccat 2161 gcgcaccttg aagcgcatga actccttgat gacgtcctcg gaggaggcca tggtggcgac 2221 cggcttcgag ccgattgttt agcttgttca gctgcgcttg tttatttgct tagctttcgc 2281 ttagcgacgt gttcactttg cttgtttgaa ttgaattgtc gctccgtaga cgaagcgcct 2341 ctatttatac tccggcggtc gagggttcga aatcgataag cttacgtaga gtctccgctc 2401 gcctgattat tttaatctcc gcctgattat tttaatgacc gcctgattat tttaatctac 2461 gcctgattat tttaaatgca tcgcctgatt attttaatct ccgcctgatt attttaatga 2521 ccgcctgatt attttaatct acgcctgatt attttaacgg gtcacggaag ttagcctagc 2581 tcgaagccgc ggtgcgggtg ccagggcgtg cccttgggct ccccgggcgc gtactccacc 2641 tcacccatct ggtccatcat gatgaacggg tcgaggtggc ggtagttgat cccggcgaac 2701 gcgcggcgca ccgggaagcc ctcgccctcg agggccccta ggctaacttc cgtgacccgt 2761 taaaataatc aggcgtagat taaaataatc aggcggtcat taaaataatc aggcggagat 2821 taaaataatc aggcgatgca tttaaaataa tcaggcgtag attaaaataa tcaggcggtc 2881 attaaaataa tcaggcggag attaaaataa tcaggcgagc ggagactcta cgtacgccgg 2941 agtataaata gaggcgcttc gtctacggag cgacaattca attcaaacaa gcaaagtgaa 3001 cacgtcgcta agcgaaagct aagcaaataa acaagcgcag ctgaacaagc taaacaatcg 3061 gacgcgtcaa catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg 3121 tcgagctgga cggcgacgta aacggccaca ggttcagcgt gtccggcgag ggcgagggcg 3181 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc 3241 cctggcccac cctcgtgacc accctgacct ggggcgtgca gtgcttcagc cgctaccccg 3301 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc 3361 gtaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg 3421 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca 3481 tcctggggca caagctggag tacaactaca tcagccacaa cgtctatatc accgccgaca 3541 agcagaagaa cggcatcaag gcccacttca agatccgcca caacatcgag gacggcagcg 3601 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc 3661 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg 3721 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc 3781 tgtacaagta aaccggtaac ttgtttattg cagcttataa tggttacaaa taaagcaata 3841 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca 3901 aactcatcaa tgtatcttag catgctcggc cggcttgtcg acgacggcgg tctccgtcgt 3961 caggatcatc cgggcactag tcgcgattgc ggccaaccgc tgggcgcggt ggtcacggtg 4021 agcacgggac gtgcgacggc gtcggcgggt gcggatacgc ggggcagcgt cagcgggttc 4081 tcgacggtca cggcgggcat gtcgacacta gttctagcca gcttttgttc cctttagtga 4141 gggttaattt cgagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat 4201 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc 4261 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga 4321 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt 4381 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg 4441 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac 4501 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg 4561 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca 4621 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc 4681 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc 4741 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag 4801 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc 4861 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca 4921 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg 4981 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg 5041 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct 5101 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa 5161 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa 5221 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa 5281 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc 5341 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga 5401 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca 5461 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc 5521 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat 5581 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc 5641 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt 5701 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc 5761 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg 5821 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt 5881 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg 5941 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga 6001 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg 6061 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg 6121 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt 6181 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc 6241 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgca //