LOCUS Exported 7766 bp ds-DNA circular SYN 13-SEP-2023 DEFINITION Sox10 promoter driving HA-tagged biotin ligase (BirA) with NLS, a Thosea asigna virus peptide (2A), and membrane-tethered mCherry protein (membCherry); flanked by Tol2 sequences. ACCESSION . VERSION . KEYWORDS pMT-sox10-NLS-BirA-2A-mCherry_Ras SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7766) AUTHORS Trinh LA, Chong-Morrison V, Gavriouchkina D, Hochgreb-Hagele T, Senanayake U, Fraser SE, Sauka-Spengler T TITLE Biotagging of Specific Cell Populations in Zebrafish Reveals Gene Regulatory Logic Encoded in the Nuclear Transcriptome. JOURNAL Cell Rep. 2017 Apr 11;19(2):425-440. doi: 10.1016/j.celrep.2017.03.045. PUBMED 28402863 REFERENCE 2 (bases 1 to 7766) AUTHORS . TITLE Direct Submission JOURNAL Exported Sep 13, 2023 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7766 /organism="synthetic DNA construct" /mol_type="other DNA" rep_origin 353..941 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 842..861 /label=pBR322ori-F /note="pBR322 origin, forward primer" primer_bind 1095..1112 /label=L4440 /note="L4440 vector, forward primer" protein_bind 1229..1250 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." promoter 1265..1295 /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 1303..1319 /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)." primer_bind 1308..1330 /label=M13/pUC Reverse /note="In lacZ gene" primer_bind 1327..1343 /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind 1327..1343 /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind 1362..1382 /label=T3 /note="T3 promoter, forward primer" promoter 1364..1382 /label=T3 promoter /note="promoter for bacteriophage T3 RNA polymerase" primer_bind 1419..1435 /label=SK primer /note="common sequencing primer, one of multiple similar variants" primer_bind 1419..1435 /label=pBluescriptSK /note="For pBluescript vector" promoter 3696..3714 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" promoter 3794..3812 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" regulatory 3874..3883 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 3997..4017 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4024..4986 /codon_start=1 /gene="birA" /product="E. coli biotin protein ligase" /label=BirA /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK" CDS 5002..5055 /codon_start=1 /product="2A peptide from Thosea asigna virus capsid protein" /label=T2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="EGRGSLLTCGDVEENPGP" CDS 5062..5769 /codon_start=1 /product="monomeric derivative of DsRed fluorescent protein (Shaner et al., 2004)" /label=mCherry /note="mammalian codon-optimized" /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA EGRHSTGGMDELYK" primer_bind complement(5207..5225) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 5475..5494 /label=mCherry-F /note="mCherry, forward primer" promoter complement(5855..5872) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(5885..5904) /label=T7 /note="T7 promoter, forward primer" promoter complement(5886..5904) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" polyA_signal 5911..6045 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(5935..5954) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 5989..6008 /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind complement(6344..6361) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(6344..6360) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(6353..6375) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 6502..6957 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(6589..6608) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 6799..6820 /label=F1ori-F /note="F1 origin, forward primer" promoter 6983..7087 /gene="bla" /label=AmpR promoter CDS 7088..182 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(7306..7325) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" ORIGIN 1 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac 61 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa 121 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt 181 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat 241 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg 301 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc 361 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg 421 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag 481 cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac ttcaagaact 541 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg 601 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc 661 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg 721 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg 781 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag 841 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc 901 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct 961 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc 1021 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc 1081 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac 1141 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 1201 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc 1261 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat 1321 ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac cctcactaaa 1381 gggaacaaaa gctggagctc caccgcggtg gcggccgctc tagaactagt ggatctgctg 1441 ggcttgctga aggtaggggg tcaagaacca gaggtgtaaa gtacttgagt aattttactt 1501 gattactgta cttaagtatt atttttgggg atttttactt tacttgagta caattaaaaa 1561 tcaatacttt tacttttact taattacatt tttttagaaa aaaaagtact ttttactcct 1621 tacaatttta tttacagtca aaaagtactt attttttgga gatcacttgg gcccggctcg 1681 agctacgtac ttaagcttga ggatcccatc gattcgaatt cctgcagccc gggggatccc 1741 cttatcagag tcaacattca tggataatga aaaaggcaat gatttttggc ttgggtgggg 1801 gggccgtcgt gagatggaaa tacaagaggt tattgttgac cagaacagcg cgcgtgtttg 1861 tgtgcgtttg gcccgtcgtt tgcctctctt ctcgtctttc tatattttct actaaagctg 1921 tttaaacgcg ttaaatgcgc tctctgtgtg tgtttatgcg cgctgatgca attataacac 1981 taacacacat agcgcgacac gatgcaggag ttcattccag tgctgcgggc ctgcgcgcgc 2041 agcatgaaca caccactgcg agcaaatctg caatattaat gaatatatat aactaaagtg 2101 tgcactttct ggtttataac tgcagcaccc cgcatacacc acagtataaa gtaatctaca 2161 atattaatga atataataaa caagaatgtt cacttttagg tttaagaatg caggcctgcg 2221 cgcgcagcat cccgcgagca caacactaga aactaaactc caatattagt taatatattc 2281 aattaaaatg tacactttct ggtttaaaac tgcaagtatg cgtgcgcagc atccgacata 2341 cccaacacta gaaactaatc tacaatattt atgaaagtat tcaactaaaa tgttcacttt 2401 ctggtttaaa actgcaagta tacgcgtgcg cagcatccga catacccaac actagaaatt 2461 aatctaaatt attaattaaa gtattcaact gaattgttca ctttctggtt taaaactgca 2521 agtatggggt gcgcagcata aaacataccc aacactagaa actaatctac aatattaatg 2581 gatgtattca actaaaatgt tcactttctg gtttaaaact gcaggaatgc gcgcgcaaca 2641 ccctgcggan actttaaaac attccgcatc agctaaaact gaaagttcag ttgatnanga 2701 tgtnncatgt tcagccatca cactgctgtt ggatgctgng ttttgtattc gggttaatat 2761 ggatgtctgt aactcaagtt caagctgaca ggtcgcatct caacagcttc tgtcgcatat 2821 aaattatttg atcgattttt ctgtaattgg acctagtcaa ccacatccac tcgtttaatt 2881 cacgattact tcacatttca gaaacacact gccatgactc tcttgatttt ttcttcttct 2941 gctggttata agtcgtcgtt tgtgctttca ccaacaaaca ctgtgtaatt cagctgccca 3001 cggcagcgcc gctccgtgtt tatccagcag cgaggcggaa tgaagccggt attattccag 3061 cactttctct cccgggagct tgaacaaacc gccaaacctt caacagtagc ttgttagcag 3121 aagtgaagct aaaataacac acacacatat acacacaccg aggcccactg acgccaccgt 3181 atgattgcag cgctgccctg gaaaaaggcg acttctaaat aaagtcaaag cctcagtgtt 3241 tgtaggtttt tcctgaacag gtttatcttg ttcatgaggt gttcgggtga acagcaaagg 3301 ggttgtgcgg tcatacagcc aaccattaaa acgacatatt aatgctattt tatacattag 3361 gaagacatgc tttgcagaag caagctgttt aataaaataa taacaatgat tattataact 3421 aataaattac ctgtgtgtgt atttttgtag gtttccatca gatctatatc cagaggaaga 3481 cggcggaagg attccttctt acaaactcga attatataaa aagaactgtt aaggtttcac 3541 tggatgatct taaataataa acaaaagcac aattatttta caagaaaaaa acatttgtaa 3601 agtataaatt aatacattta tatttaaaat aaaatttaag tgaggaaatt aaacctaccg 3661 aagtcacctg tggccgcaga actagttcta ggcctattta ggtgacacta tagaagagta 3721 caagctactt gttctttttg caggatccca tcgattcgaa ttctcggtac ctagtcgacc 3781 ttcgcgaagg cctatttagg tgacactata gaagagtaca attcaaggtt aagcggccgc 3841 aagctagatt ggcattccgg tactgttggt aaagccacca tggccaccta tgacgtccca 3901 gattacgcaa gtttgcccgg gtatgatgtt cctgattatg ctagcctccc gggttacgat 3961 gtgcccgact atgcctcact tccaggcaga tctcctccaa aaaagaagag aaaggtacga 4021 tctatgaagg ataacaccgt gccactgaaa ttgattgccc tgttagcgaa cggtgaattt 4081 cactctggcg agcagttggg tgaaacgctg ggaatgagcc gggcggctat taataaacac 4141 attcagacac tgcgtgactg gggcgttgat gtctttaccg ttccgggtaa aggatacagc 4201 ctgcctgagc ctatccagtt acttaatgct aaacagatat tgggtcagct ggatggcggt 4261 agtgtagccg tgctgccagt gattgactcc acgaatcagt accttcttga tcgtatcgga 4321 gagcttaaat cgggcgatgc ttgcattgca gaataccagc aggctggccg tggtcgccgg 4381 ggtcggaaat ggttttcgcc ttttggcgca aacttatatt tgtcgatgtt ctggcgtctg 4441 gaacaaggcc cggcggcggc gattggttta agtctggtta tcggtatcgt gatggcggaa 4501 gtattacgca agctgggtgc agataaagtt cgtgttaaat ggcctaatga cctctatctg 4561 caggatcgca agctggcagg cattctggtg gagctgactg gcaaaactgg cgatgcggcg 4621 caaatagtca ttggagccgg gatcaacatg gcaatgcgcc gtgttgaaga gagtgtcgtt 4681 aatcaggggt ggatcacgct gcaggaagcg gggatcaatc tcgatcgtaa tacgttggcg 4741 gccatgctaa tacgtgaatt acgtgctgcg ttggaactct tcgaacaaga aggattggca 4801 ccttatctgt cgcgctggga aaagctggat aattttatta atcgcccagt gaaacttatc 4861 attggtgata aagaaatatt tggcatttca cgcggaatag acaaacaggg ggctttatta 4921 cttgagcagg atggaataat aaaaccctgg atgggcggtg aaatatccct gcgtagtgca 4981 gaaaaagata tcggatcagg agagggcagg ggaagtcttc taacatgcgg ggacgtggag 5041 gaaaatcccg ggcccagatc tatggtgagc aagggcgagg aggataacat ggccatcatc 5101 aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca cgagttcgag 5161 atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa gctgaaggtg 5221 accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt catgtacggc 5281 tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct gtccttcccc 5341 gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc 5401 caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg cggcaccaac 5461 ttcccctccg acggccccgt aatgcagaag aagaccatgg gctgggaggc ctcctccgag 5521 cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct gaagctgaag 5581 gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa gcccgtgcag 5641 ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa cgaggactac 5701 accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg catggacgag 5761 ctgtacaagg gaggaggaag atctaagctg aaccctcctg atgagagtgg ccccggctgc 5821 atgagctgca agtgtgtgct ctcctgatct agaactatag tgagtcgtat tacgtatccg 5881 gactccctat agtgagtcgt attaaccggt gatccagaca tgataagata cattgatgag 5941 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat 6001 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc 6061 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttgata tcccgggttt 6121 aaacgcatgc agatctaata ctcaagtaca attttaatgg agtacttttt tacttttact 6181 caagtaagat tctagccaga tacttttact tttaattgag taaaattttc cctaagtact 6241 tgtactttca cttgagtaaa atttttgagt actttttaca cctctgtcaa gaaccatatg 6301 ccggtaccca atcgccctat aggacgtcgt attacgcgcg ctcactggcc gtcgttttac 6361 aacgtcgtga ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc 6421 ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc 6481 gcagcctgaa tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg 6541 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt 6601 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc 6661 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg 6721 gtgatggttc acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg 6781 agtccacgtt ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct 6841 cggtctattc ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg 6901 agctgattta acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg 6961 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 7021 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 7081 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 7141 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 7201 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 7261 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt 7321 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 7381 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag 7441 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac 7501 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 7561 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 7621 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 7681 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact 7741 tctgcgctcg gcccttccgg ctggct //