LOCUS Exported 5697 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION MCS for cloning promoter to drive HA-tagged biotin ligase (BirA), a Thosea asigna virus peptide (2A), and membrane-tethered mCherry protein (membCherry); flanked by Tol2 sequences. ACCESSION . VERSION . KEYWORDS pMT-MCS-cytoBirA-2A-mCherry_Ras SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 5697) 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 5697) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..5697 /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 1752..1770 /label=SP6 promoter /note="promoter for bacteriophage SP6 RNA polymerase" primer_bind 1752..1769 /label=SP6 /note="SP6 promoter, forward primer" regulatory 1832..1841 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1955..2917 /codon_start=1 /gene="birA" /product="E. coli biotin protein ligase" /label=BirA /translation="MKDNTVPLKLIALLANGEFHSGEQLGETLGMSRAAINKHIQTLRD WGVDVFTVPGKGYSLPEPIQLLNAKQILGQLDGGSVAVLPVIDSTNQYLLDRIGELKSG DACIAEYQQAGRGRRGRKWFSPFGANLYLSMFWRLEQGPAAAIGLSLVIGIVMAEVLRK LGADKVRVKWPNDLYLQDRKLAGILVELTGKTGDAAQIVIGAGINMAMRRVEESVVNQG WITLQEAGINLDRNTLAAMLIRELRAALELFEQEGLAPYLSRWEKLDNFINRPVKLIIG DKEIFGISRGIDKQGALLLEQDGIIKPWMGGEISLRSAEK" CDS 2933..2986 /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 2993..3700 /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(3138..3156) /label=mCherry-R /note="mCherry, reverse primer" primer_bind 3406..3425 /label=mCherry-F /note="mCherry, forward primer" promoter complement(3786..3803) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" primer_bind complement(3816..3835) /label=T7 /note="T7 promoter, forward primer" promoter complement(3817..3835) /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" polyA_signal 3842..3976 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3866..3885) /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind 3920..3939 /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind complement(4275..4292) /label=M13 Forward /note="In lacZ gene. Also called M13-F20 or M13 (-21) Forward" primer_bind complement(4275..4291) /label=M13 fwd /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4284..4306) /label=M13/pUC Forward /note="In lacZ gene" rep_origin 4433..4888 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(4520..4539) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 4730..4751 /label=F1ori-F /note="F1 origin, forward primer" promoter 4914..5018 /gene="bla" /label=AmpR promoter CDS 5019..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(5237..5256) /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 ctcggtacct agtcgacctt 1741 cgcgaaggcc tatttaggtg acactataga agagtacaat tcaaggttaa gcggccgcaa 1801 gctagattgg cattccggta ctgttggtaa agccaccatg gccacctatg acgtcccaga 1861 ttacgcaagt ttgcccgggt atgatgttcc tgattatgct agcctcccgg gttacgatgt 1921 gcccgactat gcctcacttc caggcagatc tcctatgaag gataacaccg tgccactgaa 1981 attgattgcc ctgttagcga acggtgaatt tcactctggc gagcagttgg gtgaaacgct 2041 gggaatgagc cgggcggcta ttaataaaca cattcagaca ctgcgtgact ggggcgttga 2101 tgtctttacc gttccgggta aaggatacag cctgcctgag cctatccagt tacttaatgc 2161 taaacagata ttgggtcagc tggatggcgg tagtgtagcc gtgctgccag tgattgactc 2221 cacgaatcag taccttcttg atcgtatcgg agagcttaaa tcgggcgatg cttgcattgc 2281 agaataccag caggctggcc gtggtcgccg gggtcggaaa tggttttcgc cttttggcgc 2341 aaacttatat ttgtcgatgt tctggcgtct ggaacaaggc ccggcggcgg cgattggttt 2401 aagtctggtt atcggtatcg tgatggcgga agtattacgc aagctgggtg cagataaagt 2461 tcgtgttaaa tggcctaatg acctctatct gcaggatcgc aagctggcag gcattctggt 2521 ggagctgact ggcaaaactg gcgatgcggc gcaaatagtc attggagccg ggatcaacat 2581 ggcaatgcgc cgtgttgaag agagtgtcgt taatcagggg tggatcacgc tgcaggaagc 2641 ggggatcaat ctcgatcgta atacgttggc ggccatgcta atacgtgaat tacgtgctgc 2701 gttggaactc ttcgaacaag aaggattggc accttatctg tcgcgctggg aaaagctgga 2761 taattttatt aatcgcccag tgaaacttat cattggtgat aaagaaatat ttggcatttc 2821 acgcggaata gacaaacagg gggctttatt acttgagcag gatggaataa taaaaccctg 2881 gatgggcggt gaaatatccc tgcgtagtgc agaaaaagat atcggatcag gagagggcag 2941 gggaagtctt ctaacatgcg gggacgtgga ggaaaatccc gggcccagat ctatggtgag 3001 caagggcgag gaggataaca tggccatcat caaggagttc atgcgcttca aggtgcacat 3061 ggagggctcc gtgaacggcc acgagttcga gatcgagggc gagggcgagg gccgccccta 3121 cgagggcacc cagaccgcca agctgaaggt gaccaagggt ggccccctgc ccttcgcctg 3181 ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc tacgtgaagc accccgccga 3241 catccccgac tacttgaagc tgtccttccc cgagggcttc aagtgggagc gcgtgatgaa 3301 cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc tccctgcagg acggcgagtt 3361 catctacaag gtgaagctgc gcggcaccaa cttcccctcc gacggccccg taatgcagaa 3421 gaagaccatg ggctgggagg cctcctccga gcggatgtac cccgaggacg gcgccctgaa 3481 gggcgagatc aagcagaggc tgaagctgaa ggacggcggc cactacgacg ctgaggtcaa 3541 gaccacctac aaggccaaga agcccgtgca gctgcccggc gcctacaacg tcaacatcaa 3601 gttggacatc acctcccaca acgaggacta caccatcgtg gaacagtacg aacgcgccga 3661 gggccgccac tccaccggcg gcatggacga gctgtacaag ggaggaggaa gatctaagct 3721 gaaccctcct gatgagagtg gccccggctg catgagctgc aagtgtgtgc tctcctgatc 3781 tagaactata gtgagtcgta ttacgtatcc ggactcccta tagtgagtcg tattaaccgg 3841 tgatccagac atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg 3901 aaaaaaatgc tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag 3961 ctgcaataaa caagttaaca acaacaattg cattcatttt atgtttcagg ttcaggggga 4021 ggtgtgggag gttttttgat atcccgggtt taaacgcatg cagatctaat actcaagtac 4081 aattttaatg gagtactttt ttacttttac tcaagtaaga ttctagccag atacttttac 4141 ttttaattga gtaaaatttt ccctaagtac ttgtactttc acttgagtaa aatttttgag 4201 tactttttac acctctgtca agaaccatat gccggtaccc aatcgcccta taggacgtcg 4261 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt 4321 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag 4381 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc 4441 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt 4501 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc 4561 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta 4621 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc 4681 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg 4741 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt 4801 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat 4861 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa 4921 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac 4981 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg 5041 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc 5101 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg 5161 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga 5221 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc 5281 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag 5341 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga 5401 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg 5461 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga 5521 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt 5581 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact 5641 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggct //