LOCUS Exported 7581 bp ds-DNA circular SYN 29-MAR-2022 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS Ac5-STABLE2-neo SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 7581) AUTHORS Gonzalez, Monika; Martin-Ruiz, Itziar; Jimenez, Silvia; Pirone, TITLE Generation of stable Drosophila cell lines using multicistronic vectors JOURNAL Scientific Reports 1, Article number: 75 REFERENCE 2 (bases 1 to 7581) AUTHORS . TITLE Direct Submission JOURNAL Exported Mar 29, 2022 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..7581 /organism="synthetic DNA construct" /mol_type="other DNA" primer_bind complement(977..995) /label=pBRforEco /note="pBR322 vectors, upsteam of EcoRI site, forward primer" promoter 1063..1167 /gene="bla" /label=AmpR promoter CDS 1168..2028 /codon_start=1 /gene="bla" /product="beta-lactamase" /label=AmpR /note="confers resistance to ampicillin, carbenicillin, and related antibiotics" /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS LIKHW" primer_bind complement(1386..1405) /label=Amp-R /note="Ampicillin resistance gene, reverse primer" rep_origin 2199..2787 /direction=RIGHT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind 2688..2707 /label=pBR322ori-F /note="pBR322 origin, forward primer" polyA_signal 3388..3522 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3425..3444) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 3479..3498 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" CDS complement(3530..4333) /codon_start=1 /gene="aph(3')-II (or nptII)" /product="aminoglycoside phosphotransferase from Tn5" /label=NeoR/KanR /note="confers resistance to neomycin, kanamycin, and G418 (Geneticin(R))" /translation="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE EHQGLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3641..3660) /label=Neo-F /note="Neomycin resistance gene, forward primer" primer_bind 4251..4270 /label=Neo-R /note="Neomycin resistance gene, reverse primer" CDS complement(4340..4393) /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 complement(4397..5113) /codon_start=1 /product="the original enhanced GFP (Yang et al., 1996)" /label=EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKGD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(4439..4460) /label=EGFP-C /note="EGFP, forward primer" primer_bind 5047..5068 /label=EGFP-N /note="EGFP, reverse primer" regulatory 5110..5119 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS complement(5138..5191) /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" primer_bind 5745..5763 /label=mCherry-R /note="mCherry, reverse primer" CDS complement(5912..5935) /codon_start=1 /product="FLAG(R) epitope tag, followed by an enterokinase cleavage site" /label=FLAG /translation="DYKDDDDK" regulatory 5935..5944 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" promoter complement(5985..913) /label=Ac5 promoter /note="Drosophila melanogaster actin 5C promoter" ORIGIN 1 cgccacgccc cgccccaccc ttccttccgc tgccccgcac ccctttcctt ccactaccgt 61 tttgccgcat ttggtattgc catctcgctt ttttggcttt actttgcttt ttgaataagc 121 caagaaactg aaatcaaagc ggacagtccg aaaattgaat attccaaaaa acaacaattg 181 cacatgcact gcgttcggca aaagacgcag aaattcaagc cttttggtct ggcatcatct 241 taaaatatca tcagtatcat cattgccatt ataaatacat aagaaaatga gaatacatta 301 aaggtatata catacaatct ttgtcatagt caacatattt tgcaacaaaa ttatctatag 361 ataacatgtt tttgtagaaa attccactaa ctgattttcg tataaataca aattattctc 421 attcttcaaa atactcgccg acgtcatcgc gaaattcctc agcattcttc gacaaatgaa 481 gagagacagc gagtgacaca atcttatctt gccgacgagt ctttgaaaat ggccccgggt 541 aacggtgaaa aacatatagc caagaatgga gcgatcgcca ggcgatggaa attggcttca 601 aattgctccg aatatggcca gttggtcagt tggcattgga ttgtggagag atactagatc 661 agctcgttct ccttctcctc cgcattctcc tactccgccc gaagacgctt ctttgatcgc 721 ttctttctcg aatgtgtaca tattcattat gtatttggtg tttggcgacg atcagccagc 781 tttaatggcc ataaaacgtg tcttggtttg tgggccacat aacaggcagc gagatctgga 841 aaaatgatgt gacagtggaa atgagaagta ggtgcatctg caaagatttg attcagagtt 901 gatgccattc atgatttttt tttagaagta tcatttgtgt ttttagaata tagaattaat 961 tcttgaagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata 1021 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt 1081 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg 1141 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt 1201 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta 1261 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc 1321 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa 1381 gttctgctat gtggcgcggt attatcccgt gttgacgccg ggcaagagca actcggtcgc 1441 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt 1501 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact 1561 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac 1621 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata 1681 ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg caacaacgtt gcgcaaacta 1741 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg 1801 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat 1861 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt 1921 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga 1981 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa 2041 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag 2101 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac 2161 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc 2221 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat 2281 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat 2341 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct 2401 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt 2461 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg 2521 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta 2581 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg 2641 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg 2701 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc 2761 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg 2821 gccttttgct ggccttgaag ctgtccctga tggtcgtcat ctacctgcct ggacagcatg 2881 gcctgcaacg cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaaggcc 2941 atccagcctc gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg 3001 gcgataatgg cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga 3061 gcgagggcgt gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag 3121 cgaaagcggt cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc 3181 atgataaaga agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag 3241 gagctgactg ggttgaaggc tctcaagggc atcggtcgac tgatcataat cagccatacc 3301 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa 3361 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa 3421 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt 3481 ggtttgtcca aactcatcaa tgtatcttat catgtctgga tccctcgagt cagaagaact 3541 cgtcaagaag gcgatagaag gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca 3601 cgaggaagcg gtcagcccat tcgccgccaa gttcttcagc aatatcacgg gtagccaacg 3661 ctatgtcctg atagcggtcc gccacaccca gccggccaca gtcgatgaat ccagaaaagc 3721 ggccattttc caccatgata ttcggcaagc aggcatcgcc atgggtcacg acgagatcct 3781 cgccgtcggg catgctcgcc ttgagcctgg cgaacagttc ggctggcgcg agcccctgat 3841 gttcttcgtc cagatcatcc tgatcgacaa gaccggcttc catccgagta cgtgctcgct 3901 cgatgcgatg tttcgcttgg tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc 3961 gccgcattgc atcagccatg atggatactt tctcggcagg agcaaggtga gatgacagga 4021 gatcctgccc cggcacttcg cccaatagca gccagtccct tcccgcttca gtgacaacgt 4081 cgagcacagc tgcgcaagga acgcccgtcg tggccagcca cgatagccgc gctgcctcgt 4141 cttgcagttc attcagggca ccggacaggt cggtcttgac aaaaagaacc gggcgcccct 4201 gcgctgacag ccggaacacg gcggcatcag agcagccgat tgtctgttgt gcccagtcat 4261 agccgaatag cctctccacc caagcggccg gagaacctgc gtgcaatcca tcttgttcaa 4321 tggccgatcc catgctagca gggccgggat tctcctccac gtcaccgcat gttagaagac 4381 ttcctctgcc ctcaagcttg tacagctcgt ccatgccgag agtgatcccg gcggcggtca 4441 cgaactccag caggaccatg tgatcgcgct tctcgttggg gtctttgctc agggcggact 4501 gggtgctcag gtagtggttg tcgggcagca gcacggggcc gtcgccgatg ggggtgttct 4561 gctggtagtg gtcggcgagc tgcacgctgc cgtcctcgat gttgtggcgg atcttgaagt 4621 tcaccttgat gccgttcttc tgcttgtcgg ccatgatata gacgttgtgg ctgttgtagt 4681 tgtactccag cttgtgcccc aggatgttgc cgtcctcctt gaagtcgatg cccttcagct 4741 cgatgcggtt caccagggtg tcgccctcga acttcacctc ggcgcgggtc ttgtagttgc 4801 cgtcgccctt gaagaagatg gtgcgctcct ggacgtagcc ttcgggcatg gcggacttga 4861 agaagtcgtg ctgcttcatg tggtcggggt agcggctgaa gcactgcacg ccgtaggtca 4921 gggtggtcac gagggtgggc cagggcacgg gcagcttgcc ggtggtgcag atgaacttca 4981 gggtcagctt gccgtaggtg gcatcgccct cgccctcgcc ggacacgctg aacttgtggc 5041 cgtttacgtc gccgtccagc tcgaccagga tgggcaccac cccggtgaac agctcctcgc 5101 ccttgctcac catggtggct ctagagatat cggaaccagg gccagggttc tcttcgacat 5161 ctccgcaagt cagtaggctg ccgcgtcctt cgcggccgcc tttatacagc tcgtccatgc 5221 cgccggtgct gtgtctgccc tcggcccgct cgtactgctc cacgatggtg tagtcctcgt 5281 tgtggctggt gatgtccagc ttgatgttca cgttgtaggc gccaggcagc tgcacgggtt 5341 tcttggcctt gtaggtggtt ttcacctcgg cgtcgtagtg gccgccatcc ttcagcttca 5401 gccgctgctt gatctcgccc ttcagggcgc catcctcggg gtacatccgc tcgctgctgg 5461 cctcccagcc catggtcttt ttctgcatca cggggccgtc gctggggaag ttggtgcccc 5521 gcagtttcac cttgtagatg aactcgccgt cctgcaggct gctgtcctgg gtcacggtca 5581 ccacgccgcc gtcctcgaag ttcatcaccc gctcccattt gaatccctcg gggaaggaca 5641 gcttcaggta gtcggggatg tcggcggggt gcttcacgta ggccttgctg ccgtacatga 5701 actgggggct caggatgtcc caggcgaagg gcagaggtcc gcccttggtc accttcagct 5761 tggcggtctg ggtgccctcg tagggcctgc cttcgccctc gccctcgatc tcgaactcgt 5821 ggccgttcac gctgccttcc atgtgcacct tgaaccgcat gaactccttg atgatggcca 5881 tgttgtcctc ttcgcccttg gacacgctgc ccttgtcgtc atcgtctttg tagtccatgg 5941 tggcgaattc caccacactg gactagtagg taccccgatc cggggtctct ggattagacg 6001 actgctggct gatggagcgg ctttgtgtcg ggaggagtat ccacacagca caagaactca 6061 aacggtagtg atatgaaaac tggtcccgca gcccgtttta taaagcccgc ttagcgctaa 6121 aaccttatca gccgcaatgc tctccgctct caagtcgcgt tcaaaacttt taccatatat 6181 gggtatgaat tgttcgccga ttgggttttc atttacggag aatttcctcc gcaactgggt 6241 gttcgtgctt gtgtgtgtct tgtatatcgc catgaagagt gtacttccat gggtcctatc 6301 agttgcggca tgtgattcaa tttgggggtt actcatattc gatacttcag ctggttatct 6361 ttatgctgtg atggtcacgg tgcttttaca cattttttcc ccaactactc attgtatgcc 6421 aaaacataca tatgtacatg tgtttatagt gggtttatta gtaatgtcca gcgatattaa 6481 aacattacag ttaaataata ttcatattag cttacgttta ctgctatctg cacttcaaat 6541 tggttattcc agggacattt tgtgcaacac acatcttcta gtagggcatt cactagtcgt 6601 agaaaacaca cctaacttaa gccggtttta acaataacgg cgaaacagct gccagacgag 6661 agaaaagtac acagcaagaa gcatttctat aatgatgccg tcctaaatat gcagcatttt 6721 tgctcaaatt gatcatttcg ttgcatcgct caggtggtat agtatttggc tattgaatgt 6781 gattgctatc ttaatagttt tgaatagtaa tagtgcaaat attcattcat aaaaaatagg 6841 taatcgattt tttcagtgca gtctatgcgt ttgcgcaaca gagacgaaga gagagacgcc 6901 tcacacacaa gcacatgcgg tctaagctgc tctccctctt tttctctctc tctctggctt 6961 gcggcagttt tccagaggct gtgactcagt aagcatttct tgttgttacc ctaacgataa 7021 gcgtccgatt cctaattctc tttcaaataa tttgttttat tttttttttt gtgtgttttg 7081 aagtcaaaca gcttcataaa caatattaaa tatcgcaatt gttattgcaa atgcattcgt 7141 atcaggcgcc ttgatgtttt tcgccaaaac ttcgtctgct ctttgtcagc gtaattgcgt 7201 ttgccggtcg tgccgtcttg ctcgcacgcg gcttgcaaac aaaaagtgaa agagggaaag 7261 ccacctgtgt tgttgctttt gttgctatcg ttggctgcac acacatggca ataagtaacc 7321 atattgtaga cttatttatt ttagcgtttt cgttgactaa tctttgttta aataattgcc 7381 tatttcgagc caaaatgacg cataaaagga aacggcaaca tgcggtgcgt tgagcatgcg 7441 taagcgagat ggcgctagag agagcgcagc ataaaaagaa aaaaacaacc ggcgagtggc 7501 gagacacgaa taatacacac acgcacacat ggtcaaagga gtggctccaa cgtcggtgac 7561 gtcgcgccca gccacagacc c //