LOCUS Exported 6455 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION synthetic circular DNA ACCESSION . VERSION . KEYWORDS pcDNA3.3_d2eGFP SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6455) AUTHORS Warren L, Manos PD, Ahfeldt T, Loh YH, Li H, Lau F, Ebina W, Mandal PK, Smith ZD, Meissner A, Daley GQ, Brack AS, Collins JJ, Cowan C, Schlaeger TM, Rossi DJ TITLE Highly Efficient Reprogramming to Pluripotency and Directed Differentiation of Human Cells with Synthetic Modified mRNA. JOURNAL Cell Stem Cell. 2010 Sep 29. ():. PUBMED 20888316 REFERENCE 2 (bases 1 to 6455) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6455 /organism="synthetic DNA construct" /mol_type="other DNA" enhancer 50..429 /label=CMV enhancer /note="human cytomegalovirus immediate early enhancer" promoter 430..633 /label=CMV promoter /note="human cytomegalovirus (CMV) immediate early promoter" primer_bind 584..604 /label=CMV-F /note="Human CMV immediate early promoter, forward primer" primer_bind 630..654 /label=LNCX /note="Human CMV promoter, forward primer" primer_bind 761..780 /label=T7 /note="T7 promoter, forward primer" promoter 761..779 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" regulatory 819..828 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 825..1670 /codon_start=1 /product="EGFP destabilized by fusion to residues 422-461 of mouse ornithine decarboxylase, giving an in vivo half-life of ~2 hours" /label=d2EGFP /note="mammalian codon-optimized" /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYKKLSHGFPPEVEEQDDGTLPMSCAQESGMDRHPAACASARINV " primer_bind complement(870..891) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(1131..1150) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 1478..1499 /label=EGFP-C /note="EGFP, forward primer" primer_bind complement(1841..1860) /label=TK-pA-R /note="Thymidine kinase polyA, reverse primer" polyA_signal 1885..1933 /label=HSV TK poly(A) signal /note="herpes simplex virus thymidine kinase polyadenylation signal (Cole and Stacy, 1985)" rep_origin 2135..2563 /direction=RIGHT /label=f1 ori /note="f1 bacteriophage origin of replication; arrow indicates direction of (+) strand synthesis" primer_bind complement(2222..2241) /label=F1ori-R /note="F1 origin, reverse primer" primer_bind 2432..2453 /label=F1ori-F /note="F1 origin, forward primer" primer_bind complement(2572..2592) /label=pBABE 3' /note="SV40 enhancer, reverse primer for pBABE vectors" promoter 2577..2906 /label=SV40 promoter /note="SV40 enhancer and early promoter" rep_origin 2757..2892 /label=SV40 ori /note="SV40 origin of replication" primer_bind 2819..2838 /label=SV40pro-F /note="SV40 promoter/origin, forward primer" CDS 2973..3767 /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 GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF" primer_bind complement(3027..3046) /label=Neo-R /note="Neomycin resistance gene, reverse primer" primer_bind 3637..3656 /label=Neo-F /note="Neomycin resistance gene, forward primer" polyA_signal 3943..4064 /label=SV40 poly(A) signal /note="SV40 polyadenylation signal" primer_bind complement(3980..3999) /label=SV40pA-R /note="SV40 polyA, reverse primer" primer_bind 4034..4053 /label=EBV-rev /note="SV40 polyA terminator, reverse primer" primer_bind complement(4113..4129) /label=M13 rev /note="common sequencing primer, one of multiple similar variants" primer_bind complement(4113..4129) /label=M13 Reverse /note="In lacZ gene. Also called M13-rev" primer_bind complement(4126..4148) /label=M13/pUC Reverse /note="In lacZ gene" protein_bind 4137..4153 /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)." promoter complement(4161..4191) /label=lac promoter /note="promoter for the E. coli lac operon" protein_bind 4206..4227 /label=CAP binding site /bound_moiety="E. coli catabolite activator protein" /note="CAP binding activates transcription in the presence of cAMP." primer_bind complement(4344..4361) /label=L4440 /note="L4440 vector, forward primer" rep_origin complement(4515..5103) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(4595..4614) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(5274..6134) /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 5897..5916 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(6135..6239) /gene="bla" /label=AmpR promoter primer_bind complement(6314..6333) /label=pRS-marker /note="pRS vectors, use to sequence yeast selectable marker" ORIGIN 1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta 61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag 121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc 181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga 241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat 301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc 361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct 421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca 481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat 541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg 601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg 661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg 721 actctagagg atcgaaccct ttggaccctc gtacagaagc taatacgact cactataggg 781 aaataagaga gaaaagaaga gtaagaagaa atataagagc caccatggtg agcaagggcg 841 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc 901 acaagttcag cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga 961 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga 1021 cctacggcgt gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca 1081 agtccgccat gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca 1141 actacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc 1201 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact 1261 acaacagcca caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact 1321 tcaagatccg ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga 1381 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt 1441 ccgccctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga 1501 ccgccgccgg gatcactctc ggcatggacg agctgtacaa gaagcttagc catggcttcc 1561 cgccggaggt ggaggagcag gatgatggca cgctgcccat gtcttgtgcc caggagagcg 1621 ggatggaccg tcaccctgca gcctgtgctt ctgctaggat caatgtgtag gctgccttct 1681 gcggggcttg ccttctggcc atgcccttct tctctccctt gcacctgtac ctcttggtct 1741 ttgaataaag cctgagtagg aagtgagggt ctagaactag tgtcgacgca agggttcgat 1801 ccctaccggt tagtaatgag tttaaacggg ggaggctaac tgaaacacgg aaggagacaa 1861 taccggaagg aacccgcgct atgacggcaa taaaaagaca gaataaaacg cacgggtgtt 1921 gggtcgtttg ttcataaacg cggggttcgg tcccagggct ggcactctgt cgatacccca 1981 ccgagacccc attggggcca atacgcccgc gtttcttcct tttccccacc ccacccccca 2041 agttcgggtg aaggcccagg gctcgcagcc aacgtcgggg cggcaggccc tgccatagca 2101 gatctgcgca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc 2161 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc 2221 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct 2281 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa 2341 aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc 2401 cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca 2461 ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat 2521 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt 2581 gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc 2641 atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta 2701 tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc 2761 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta 2821 tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct 2881 tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat 2941 ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag 3001 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg 3061 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca 3121 agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg ctatcgtggc 3181 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg 3241 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg 3301 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta 3361 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag 3421 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac 3481 tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg acccatggcg 3541 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg 3601 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg 3661 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg 3721 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg 3781 gttcgcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc 3841 gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc 3901 ctccagcgcg gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct 3961 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca 4021 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg 4081 tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt 4141 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt 4201 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg 4261 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg 4321 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg 4381 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat 4441 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc 4501 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc 4561 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga 4621 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt 4681 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg 4741 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc 4801 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg 4861 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc 4921 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg 4981 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc 5041 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct 5101 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt 5161 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa 5221 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa 5281 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc 5341 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct 5401 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca 5461 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt 5521 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt 5581 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc 5641 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc 5701 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt 5761 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact 5821 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc 5881 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt 5941 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg 6001 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct 6061 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa 6121 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt 6181 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc 6241 acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatctccc gatcccctat 6301 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg 6361 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag 6421 gcttgaccga caattgcatg aagaatctgc ttagg //