LOCUS Exported 8360 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION AdEasy Shuttle vector modified for multicistronic expression. Contains T2A, F2A and Venus tag. ACCESSION . VERSION . KEYWORDS pShuttle-CMV-F2A-T2A-Venus SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 8360) TITLE Modified AdEasy shuttle vectors for multicistronic expression JOURNAL Unpublished REFERENCE 2 (bases 1 to 8360) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..8360 /organism="synthetic DNA construct" /mol_type="other DNA" repeat_region 1..103 /label=ITR /note="inverted terminal repeat of human adenovirus serotype 5" misc_signal 191..341 /label=Ad5 Psi /note="packaging signal for adenovirus serotype 5" enhancer 399..702 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 703..906 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 857..877 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" CDS 969..1034 /codon_start=1 /product="2A peptide from foot-and-mouth disease virus polyprotein" /label=F2A /note="Eukaryotic ribosomes fail to insert a peptide bond between the Gly and Pro residues, yielding separate polypeptides." /translation="VKQTLNFDLLKLAGDVESNPGP" CDS 1065..1118 /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" regulatory 1125..1134 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 1131..1850 /codon_start=1 /product="yellow fluorescent protein (YFP) with fast and efficient maturation (Nagai et al., 2002)" /label=Venus /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITADKQKNGIK ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(1176..1197) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1437..1456) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1784..1805 /label=EGFP-C /note="EGFP, forward primer" polyA_signal 2008..2129 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(2045..2064) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 2099..2118 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" repeat_region 5320..5422 /label=ITR /note="inverted terminal repeat of human adenovirus serotype 5" primer_bind complement(5511..5528) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(5682..6270) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(5762..5781) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS 7102..7896 /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="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(7156..7175) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 7766..7785 /label=Neo-F /note="Neomycin resistance gene, forward primer" rep_origin complement(7944..8323) /direction=LEFT /label=M13 ori /note="M13 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(8006..8027) /label=F1ori-F /note="F1 origin, forward primer" primer_bind 8218..8237 /label=F1ori-R /note="F1 origin, reverse primer" ORIGIN 1 catcatcaat aatatacctt attttggatt gaagccaata tgataatgag ggggtggagt 61 ttgtgacgtg gcgcggggcg tgggaacggg gcgggtgacg tagtagtgtg gcggaagtgt 121 gatgttgcaa gtgtggcgga acacatgtaa gcgacggatg tggcaaaagt gacgtttttg 181 gtgtgcgccg gtgtacacag gaagtgacaa ttttcgcgcg gttttaggcg gatgttgtag 241 taaatttggg cgtaaccgag taagatttgg ccattttcgc gggaaaactg aataagagga 301 agtgaaatct gaataatttt gtgttactca tagcgcgtaa tactgtaata gtaatcaatt 361 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat 421 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt 481 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa 541 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc 601 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct 661 acttggcagt acatctacgt attagtcatc gctattacca tggtgatgcg gttttggcag 721 tacatcaatg ggcgtggata gcggtttgac tcacggggat ttccaagtct ccaccccatt 781 gacgtcaatg ggagtttgtt ttggcaccaa aatcaacggg actttccaaa atgtcgtaac 841 aactccgccc cattgacgca aatgggcggt aggcgtgtac ggtgggaggt ctatataagc 901 agagctggtt tagtgaaccg tcagatccgc tagagatctg gtaccgtcga cgcggccgcg 961 gaagcggagt gaaacagact ttgaattttg accttctcaa gttggcggga gacgtggagt 1021 ccaaccctgg acctaggcct ggaagcggaa cgcgtggaag cggagagggc agaggaagtc 1081 tgctaacatg cggtgacgtc gaggagaatc ctggacctct cgaggccacc atggtgagca 1141 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa 1201 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga 1261 ccctgaagct gatctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca 1321 ccctgggcta cggcctgcag tgcttcgccc gctaccccga ccacatgaag cagcacgact 1381 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg 1441 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca 1501 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt 1561 acaactacaa cagccacaac gtctatatca ccgccgacaa gcagaagaac ggcatcaagg 1621 ccaacttcaa gatccgccac aacatcgagg acggcggcgt gcagctcgcc gaccactacc 1681 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagct 1741 accagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt 1801 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtag taatgagcaa 1861 gcttctagat aagatatccg atccaccgga tctagataac tgatcataat cagccatacc 1921 acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct gaacctgaaa 1981 cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa tggttacaaa 2041 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt 2101 ggtttgtcca aactcatcaa tgtatcttaa cgcggatctg ggcgtggtta agggtgggaa 2161 agaatatata aggtgggggt cttatgtagt tttgtatctg ttttgcagca gccgccgccg 2221 ccatgagcac caactcgttt gatggaagca ttgtgagctc atatttgaca acgcgcatgc 2281 ccccatgggc cggggtgcgt cagaatgtga tgggctccag cattgatggt cgccccgtcc 2341 tgcccgcaaa ctctactacc ttgacctacg agaccgtgtc tggaacgccg ttggagactg 2401 cagcctccgc cgccgcttca gccgctgcag ccaccgcccg cgggattgtg actgactttg 2461 ctttcctgag cccgcttgca agcagtgcag cttcccgttc atccgcccgc gatgacaagt 2521 tgacggctct tttggcacaa ttggattctt tgacccggga acttaatgtc gtttctcagc 2581 agctgttgga tctgcgccag caggtttctg ccctgaaggc ttcctcccct cccaatgcgg 2641 tttaaaacat aaataaaaaa ccagactctg tttggatttg gatcaagcaa gtgtcttgct 2701 gtctttattt aggggttttg cgcgcgcggt aggcccggga ccagcggtct cggtcgttga 2761 gggtcctgtg tattttttcc aggacgtggt aaaggtgact ctggatgttc agatacatgg 2821 gcataagccc gtctctgggg tggaggtagc accactgcag agcttcatgc tgcggggtgg 2881 tgttgtagat gatccagtcg tagcaggagc gctgggcgtg gtgcctaaaa atgtctttca 2941 gtagcaagct gattgccagg ggcaggccct tggtgtaagt gtttacaaag cggttaagct 3001 gggatgggtg catacgtggg gatatgagat gcatcttgga ctgtattttt aggttggcta 3061 tgttcccagc catatccctc cggggattca tgttgtgcag aaccaccagc acagtgtatc 3121 cggtgcactt gggaaatttg tcatgtagct tagaaggaaa tgcgtggaag aacttggaga 3181 cgcccttgtg acctccaaga ttttccatgc attcgtccat aatgatggca atgggcccac 3241 gggcggcggc ctgggcgaag atatttctgg gatcactaac gtcatagttg tgttccagga 3301 tgagatcgtc ataggccatt tttacaaagc gcgggcggag ggtgccagac tgcggtataa 3361 tggttccatc cggcccaggg gcgtagttac cctcacagat ttgcatttcc cacgctttga 3421 gttcagatgg ggggatcatg tctacctgcg gggcgatgaa gaaaacggtt tccggggtag 3481 gggagatcag ctgggaagaa agcaggttcc tgagcagctg cgacttaccg cagccggtgg 3541 gcccgtaaat cacacctatt accggctgca actggtagtt aagagagctg cagctgccgt 3601 catccctgag caggggggcc acttcgttaa gcatgtccct gactcgcatg ttttccctga 3661 ccaaatccgc cagaaggcgc tcgccgccca gcgatagcag ttcttgcaag gaagcaaagt 3721 ttttcaacgg tttgagaccg tccgccgtag gcatgctttt gagcgtttga ccaagcagtt 3781 ccaggcggtc ccacagctcg gtcacctgct ctacggcatc tcgatccagc atatctcctc 3841 gtttcgcggg ttggggcggc tttcgctgta cggcagtagt cggtgctcgt ccagacgggc 3901 cagggtcatg tctttccacg ggcgcagggt cctcgtcagc gtagtctggg tcacggtgaa 3961 ggggtgcgct ccgggctgcg cgctggccag ggtgcgcttg aggctggtcc tgctggtgct 4021 gaagcgctgc cggtcttcgc cctgcgcgtc ggccaggtag catttgacca tggtgtcata 4081 gtccagcccc tccgcggcgt ggcccttggc gcgcagcttg cccttggagg aggcgccgca 4141 cgaggggcag tgcagacttt tgagggcgta gagcttgggc gcgagaaata ccgattccgg 4201 ggagtaggca tccgcgccgc aggccccgca gacggtctcg cattccacga gccaggtgag 4261 ctctggccgt tcggggtcaa aaaccaggtt tcccccatgc tttttgatgc gtttcttacc 4321 tctggtttcc atgagccggt gtccacgctc ggtgacgaaa aggctgtccg tgtccccgta 4381 tacagacttg agagggagtt taaacgaatt caatagcttg ttgcatgggc ggcgatataa 4441 aatgcaaggt gctgctcaaa aaatcaggca aagcctcgcg caaaaaagaa agcacatcgt 4501 agtcatgctc atgcagataa aggcaggtaa gctccggaac caccacagaa aaagacacca 4561 tttttctctc aaacatgtct gcgggtttct gcataaacac aaaataaaat aacaaaaaaa 4621 catttaaaca ttagaagcct gtcttacaac aggaaaaaca acccttataa gcataagacg 4681 gactacggcc atgccggcgt gaccgtaaaa aaactggtca ccgtgattaa aaagcaccac 4741 cgacagctcc tcggtcatgt ccggagtcat aatgtaagac tcggtaaaca catcaggttg 4801 attcacatcg gtcagtgcta aaaagcgacc gaaatagccc gggggaatac atacccgcag 4861 gcgtagagac aacattacag cccccatagg aggtataaca aaattaatag gagagaaaaa 4921 cacataaaca cctgaaaaac cctcctgcct aggcaaaata gcaccctccc gctccagaac 4981 aacatacagc gcttccacag cggcagccat aacagtcagc cttaccagta aaaaagaaaa 5041 cctattaaaa aaacaccact cgacacggca ccagctcaat cagtcacagt gtaaaaaagg 5101 gccaagtgca gagcgagtat atataggact aaaaaatgac gtaacggtta aagtccacaa 5161 aaaacaccca gaaaaccgca cgcgaaccta cgcccagaaa cgaaagccaa aaaacccaca 5221 acttcctcaa atcgtcactt ccgttttccc acgttacgtc acttcccatt ttaagaaaac 5281 tacaattccc aacacataca agttactccg ccctaaaacc tacgtcaccc gccccgttcc 5341 cacgccccgc gccacgtcac aaactccacc ccctcattat catattggct tcaatccaaa 5401 ataaggtata ttattgatga tgttaattaa catgcatgga tccatatgcg gtgtgaaata 5461 ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc ctcgctcact 5521 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta 5581 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag 5641 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 5701 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 5761 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 5821 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 5881 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 5941 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 6001 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 6061 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 6121 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 6181 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 6241 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 6301 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 6361 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 6421 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 6481 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 6541 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 6601 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 6661 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 6721 ccagttaata gtttgcgcaa cgttgttgcc attgctgcag ccatgagatt atcaaaaagg 6781 atcttcacct agatcctttt cacgtagaaa gccagtccgc agaaacggtg ctgaccccgg 6841 atgaatgtca gctactgggc tatctggaca agggaaaacg caagcgcaaa gagaaagcag 6901 gtagcttgca gtgggcttac atggcgatag ctagactggg cggttttatg gacagcaagc 6961 gaaccggaat tgccagctgg ggcgccctct ggtaaggttg ggaagccctg caaagtaaac 7021 tggatggctt tcttgccgcc aaggatctga tggcgcaggg gatcaagctc tgatcaagag 7081 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc 7141 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat 7201 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg 7261 tccggtgccc tgaatgaact gcaagacgag gcagcgcggc tatcgtggct ggccacgacg 7321 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta 7381 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta 7441 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc 7501 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc 7561 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg 7621 ctcaaggcga gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg 7681 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt 7741 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc 7801 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc 7861 atcgccttct atcgccttct tgacgagttc ttctgaattt tgttaaaatt tttgttaaat 7921 cagctcattt tttaaccaat aggccgaaat cggcaccatc ccttataaat caaaagaata 7981 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt 8041 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc 8101 atcaccctaa tcaagttttt tgtggtcgag gtgccgtaaa gcactaaatc ggaaccctaa 8161 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg 8221 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt 8281 aaccaccaca cccgcgcgct taatgcgccg ctacagggcg cgtccattcg ccattcagga 8341 tcgaattaat tcttaattaa //