LOCUS Exported 10712 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION TC-83 I-SceI T7 5'UTR (A3G) P1234 (nsP2 Q739L) SGP XbaI attB1 Kozak EGFP-PEST ochre attB2 AscI (truncated E1) 3'UTR Poly A I-SceI. ACCESSION . VERSION . KEYWORDS pTK313 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10712) AUTHORS Wroblewska L, Kitada T, Endo K, Siciliano V, Stillo B, Saito H, Weiss R TITLE Mammalian synthetic circuits with RNA binding proteins for RNA-only delivery. JOURNAL Nat Biotechnol. 2015 Aug 3. doi: 10.1038/nbt.3301. PUBMED 26237515 REFERENCE 2 (bases 1 to 10712) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10712 /organism="synthetic DNA construct" /mol_type="other DNA" RBS 1535..1543 /label=Shine-Dalgarno sequence /note="full consensus sequence for ribosome-binding sites upstream of start codons in E. coli; complementary to a region in the 3' end of the 16S rRNA (Chen et al., 1994)" protein_bind 7568..7592 /gene="mutant version of attB" /label=attB1 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" regulatory 7593..7602 /regulatory_class="other" /note="vertebrate consensus sequence for strong initiation of translation (Kozak, 1987)" CDS 7599..8444 /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(7644..7665) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(7905..7924) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 8252..8273 /label=EGFP-C /note="EGFP, forward primer" protein_bind complement(8445..8469) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" rep_origin complement(8922..9510) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(9002..9021) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(9681..10541) /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 10304..10323 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(10542..10646) /gene="bla" /label=AmpR promoter promoter 10695..10712 /label=T7 promoter /note="promoter for bacteriophage T7 RNA polymerase" ORIGIN 1 atgggcggcg catgagagaa gcccagacca attacctacc caaaatggag aaagttcacg 61 ttgacatcga ggaagacagc ccattcctca gagctttgca gcggagcttc ccgcagtttg 121 aggtagaagc caagcaggtc actgataatg accatgctaa tgccagagcg ttttcgcatc 181 tggcttcaaa actgatcgaa acggaggtgg acccatccga cacgatcctt gacattggaa 241 gtgcgcccgc ccgcagaatg tattctaagc acaagtatca ttgtatctgt ccgatgagat 301 gtgcggaaga tccggacaga ttgtataagt atgcaactaa gctgaagaaa aactgtaagg 361 aaataactga taaggaattg gacaagaaaa tgaaggagct cgccgccgtc atgagcgacc 421 ctgacctgga aactgagact atgtgcctcc acgacgacga gtcgtgtcgc tacgaagggc 481 aagtcgctgt ttaccaggat gtatacgcgg ttgacggacc gacaagtctc tatcaccaag 541 ccaataaggg agttagagtc gcctactgga taggctttga caccacccct tttatgttta 601 agaacttggc tggagcatat ccatcatact ctaccaactg ggccgacgaa accgtgttaa 661 cggctcgtaa cataggccta tgcagctctg acgttatgga gcggtcacgt agagggatgt 721 ccattcttag aaagaagtat ttgaaaccat ccaacaatgt tctattctct gttggctcga 781 ccatctacca cgagaagagg gacttactga ggagctggca cctgccgtct gtatttcact 841 tacgtggcaa gcaaaattac acatgtcggt gtgagactat agttagttgc gacgggtacg 901 tcgttaaaag aatagctatc agtccaggcc tgtatgggaa gccttcaggc tatgctgcta 961 cgatgcaccg cgagggattc ttgtgctgca aagtgacaga cacattgaac ggggagaggg 1021 tctcttttcc cgtgtgcacg tatgtgccag ctacattgtg tgaccaaatg actggcatac 1081 tggcaacaga tgtcagtgcg gacgacgcgc aaaaactgct ggttgggctc aaccagcgta 1141 tagtcgtcaa cggtcgcacc cagagaaaca ccaataccat gaaaaattac cttttgcccg 1201 tagtggccca ggcatttgct aggtgggcaa aggaatataa ggaagatcaa gaagatgaaa 1261 ggccactagg actacgagat agacagttag tcatggggtg ttgttgggct tttagaaggc 1321 acaagataac atctatttat aagcgcccgg atacccaaac catcatcaaa gtgaacagcg 1381 atttccactc attcgtgctg cccaggatag gcagtaacac attggagatc gggctgagaa 1441 caagaatcag gaaaatgtta gaggagcaca aggagccgtc acctctcatt accgccgagg 1501 acgtacaaga agctaagtgc gcagccgatg aggctaagga ggtgcgtgaa gccgaggagt 1561 tgcgcgcagc tctaccacct ttggcagctg atgttgagga gcccactctg gaagccgatg 1621 tcgacttgat gttacaagag gctggggccg gctcagtgga gacacctcgt ggcttgataa 1681 aggttaccag ctacgatggc gaggacaaga tcggctctta cgctgtgctt tctccgcagg 1741 ctgtactcaa gagtgaaaaa ttatcttgca tccaccctct cgctgaacaa gtcatagtga 1801 taacacactc tggccgaaaa gggcgttatg ccgtggaacc ataccatggt aaagtagtgg 1861 tgccagaggg acatgcaata cccgtccagg actttcaagc tctgagtgaa agtgccacca 1921 ttgtgtacaa cgaacgtgag ttcgtaaaca ggtacctgca ccatattgcc acacatggag 1981 gagcgctgaa cactgatgaa gaatattaca aaactgtcaa gcccagcgag cacgacggcg 2041 aatacctgta cgacatcgac aggaaacagt gcgtcaagaa agaactagtc actgggctag 2101 ggctcacagg cgagctggtg gatcctccct tccatgaatt cgcctacgag agtctgagaa 2161 cacgaccagc cgctccttac caagtaccaa ccataggggt gtatggcgtg ccaggatcag 2221 gcaagtctgg catcattaaa agcgcagtca ccaaaaaaga tctagtggtg agcgccaaga 2281 aagaaaactg tgcagaaatt ataagggacg tcaagaaaat gaaagggctg gacgtcaatg 2341 ccagaactgt ggactcagtg ctcttgaatg gatgcaaaca ccccgtagag accctgtata 2401 ttgacgaagc ttttgcttgt catgcaggta ctctcagagc gctcatagcc attataagac 2461 ctaaaaaggc agtgctctgc ggggatccca aacagtgcgg tttttttaac atgatgtgcc 2521 tgaaagtgca ttttaaccac gagatttgca cacaagtctt ccacaaaagc atctctcgcc 2581 gttgcactaa atctgtgact tcggtcgtct caaccttgtt ttacgacaaa aaaatgagaa 2641 cgacgaatcc gaaagagact aagattgtga ttgacactac cggcagtacc aaacctaagc 2701 aggacgatct cattctcact tgtttcagag ggtgggtgaa gcagttgcaa atagattaca 2761 aaggcaacga aataatgacg gcagctgcct ctcaagggct gacccgtaaa ggtgtgtatg 2821 ccgttcggta caaggtgaat gaaaatcctc tgtacgcacc cacctcagaa catgtgaacg 2881 tcctactgac ccgcacggag gaccgcatcg tgtggaaaac actagccggc gacccatgga 2941 taaaaacact gactgccaag taccctggga atttcactgc cacgatagag gagtggcaag 3001 cagagcatga tgccatcatg aggcacatct tggagagacc ggaccctacc gacgtcttcc 3061 agaataaggc aaacgtgtgt tgggccaagg ctttagtgcc ggtgctgaag accgctggca 3121 tagacatgac cactgaacaa tggaacactg tggattattt tgaaacggac aaagctcact 3181 cagcagagat agtattgaac caactatgcg tgaggttctt tggactcgat ctggactccg 3241 gtctattttc tgcacccact gttccgttat ccattaggaa taatcactgg gataactccc 3301 cgtcgcctaa catgtacggg ctgaataaag aagtggtccg tcagctctct cgcaggtacc 3361 cacaactgcc tcgggcagtt gccactggaa gagtctatga catgaacact ggtacactgc 3421 gcaattatga tccgcgcata aacctagtac ctgtaaacag aagactgcct catgctttag 3481 tcctccacca taatgaacac ccacagagtg acttttcttc attcgtcagc aaattgaagg 3541 gcagaactgt cctggtggtc ggggaaaagt tgtccgtccc aggcaaaatg gttgactggt 3601 tgtcagaccg gcctgaggct accttcagag ctcggctgga tttaggcatc ccaggtgatg 3661 tgcccaaata tgacataata tttgttaatg tgaggacccc atataaatac catcactatc 3721 agcagtgtga agaccatgcc attaagctta gcatgttgac caagaaagct tgtctgcatc 3781 tgaatcccgg cggaacctgt gtcagcatag gttatggtta cgctgacagg gccagcgaaa 3841 gcatcattgg tgctatagcg cggctgttca agttttcccg ggtatgcaaa ccgaaatcct 3901 cacttgaaga gacggaagtt ctgtttgtat tcattgggta cgatcgcaag gcccgtacgc 3961 acaatcctta caagctttca tcaaccttga ccaacattta tacaggttcc agactccacg 4021 aagccggatg tgcaccctca tatcatgtgg tgcgagggga tattgccacg gccaccgaag 4081 gagtgattat aaatgctgct aacagcaaag gacaacctgg cggaggggtg tgcggagcgc 4141 tgtataagaa attcccggaa agcttcgatt tacagccgat cgaagtagga aaagcgcgac 4201 tggtcaaagg tgcagctaaa catatcattc atgccgtagg accaaacttc aacaaagttt 4261 cggaggttga aggtgacaaa cagttggcag aggcttatga gtccatcgct aagattgtca 4321 acgataacaa ttacaagtca gtagcgattc cactgttgtc caccggcatc ttttccggga 4381 acaaagatcg actaacccaa tcattgaacc atttgctgac agctttagac accactgatg 4441 cagatgtagc catatactgc agggacaaga aatgggaaat gactctcaag gaagcagtgg 4501 ctaggagaga agcagtggag gagatatgca tatccgacga ctcttcagtg acagaacctg 4561 atgcagagct ggtgagggtg catccgaaga gttctttggc tggaaggaag ggctacagca 4621 caagcgatgg caaaactttc tcatatttgg aagggaccaa gtttcaccag gcggccaagg 4681 atatagcaga aattaatgcc atgtggcccg ttgcaacgga ggccaatgag caggtatgca 4741 tgtatatcct cggagaaagc atgagcagta ttaggtcgaa atgccccgtc gaagagtcgg 4801 aagcctccac accacctagc acgctgcctt gcttgtgcat ccatgccatg actccagaaa 4861 gagtacagcg cctaaaagcc tcacgtccag aacaaattac tgtgtgctca tcctttccat 4921 tgccgaagta tagaatcact ggtgtgcaga agatccaatg ctcccagcct atattgttct 4981 caccgaaagt gcctgcgtat attcatccaa ggaagtatct cgtggaaaca ccaccggtag 5041 acgagactcc ggagccatcg gcagagaacc aatccacaga ggggacacct gaacaaccac 5101 cacttataac cgaggatgag accaggacta gaacgcctga gccgatcatc atcgaagagg 5161 aagaagagga tagcataagt ttgctgtcag atggcccgac ccaccaggtg ctgcaagtcg 5221 aggcagacat tcacgggccg ccctctgtat ctagctcatc ctggtccatt cctcatgcat 5281 ccgactttga tgtggacagt ttatccatac ttgacaccct ggagggagct agcgtgacca 5341 gcggggcaac gtcagccgag actaactctt acttcgcaaa gagtatggag tttctggcgc 5401 gaccggtgcc tgcgcctcga acagtattca ggaaccctcc acatcccgct ccgcgcacaa 5461 gaacaccgtc acttgcaccc agcagggcct gctcgagaac cagcctagtt tccaccccgc 5521 caggcgtgaa tagggtgatc actagagagg agctcgaggc gcttaccccg tcacgcactc 5581 ctagcaggtc ggtctcgaga accagcctgg tctccaaccc gccaggcgta aatagggtga 5641 ttacaagaga ggagtttgag gcgttcgtag cacaacaaca atgacggttt gatgcgggtg 5701 catacatctt ttcctccgac accggtcaag ggcatttaca acaaaaatca gtaaggcaaa 5761 cggtgctatc cgaagtggtg ttggagagga ccgaattgga gatttcgtat gccccgcgcc 5821 tcgaccaaga aaaagaagaa ttactacgca agaaattaca gttaaatccc acacctgcta 5881 acagaagcag ataccagtcc aggaaggtgg agaacatgaa agccataaca gctagacgta 5941 ttctgcaagg cctagggcat tatttgaagg cagaaggaaa agtggagtgc taccgaaccc 6001 tgcatcctgt tcctttgtat tcatctagtg tgaaccgtgc cttttcaagc cccaaggtcg 6061 cagtggaagc ctgtaacgcc atgttgaaag agaactttcc gactgtggct tcttactgta 6121 ttattccaga gtacgatgcc tatttggaca tggttgacgg agcttcatgc tgcttagaca 6181 ctgccagttt ttgccctgca aagctgcgca gctttccaaa gaaacactcc tatttggaac 6241 ccacaatacg atcggcagtg ccttcagcga tccagaacac gctccagaac gtcctggcag 6301 ctgccacaaa aagaaattgc aatgtcacgc aaatgagaga attgcccgta ttggattcgg 6361 cggcctttaa tgtggaatgc ttcaagaaat atgcgtgtaa taatgaatat tgggaaacgt 6421 ttaaagaaaa ccccatcagg cttactgaag aaaacgtggt aaattacatt accaaattaa 6481 aaggaccaaa agctgctgct ctttttgcga agacacataa tttgaatatg ttgcaggaca 6541 taccaatgga caggtttgta atggacttaa agagagacgt gaaagtgact ccaggaacaa 6601 aacatactga agaacggccc aaggtacagg tgatccaggc tgccgatccg ctagcaacag 6661 cgtatctgtg cggaatccac cgagagctgg ttaggagatt aaatgcggtc ctgcttccga 6721 acattcatac actgtttgat atgtcggctg aagactttga cgctattata gccgagcact 6781 tccagcctgg ggattgtgtt ctggaaactg acatcgcgtc gtttgataaa agtgaggacg 6841 acgccatggc tctgaccgcg ttaatgattc tggaagactt aggtgtggac gcagagctgt 6901 tgacgctgat tgaggcggct ttcggcgaaa tttcatcaat acatttgccc actaaaacta 6961 aatttaaatt cggagccatg atgaaatctg gaatgttcct cacactgttt gtgaacacag 7021 tcattaacat tgtaatcgca agcagagtgt tgagagaacg gctaaccgga tcaccatgtg 7081 cagcattcat tggagatgac aatatcgtga aaggagtcaa atcggacaaa ttaatggcag 7141 acaggtgcgc cacctggttg aatatggaag tcaagattat agatgctgtg gtgggcgaga 7201 aagcgcctta tttctgtgga gggtttattt tgtgtgactc cgtgaccggc acagcgtgcc 7261 gtgtggcaga ccccctaaaa aggctgttta agcttggcaa acctctggca gcagacgatg 7321 aacatgatga tgacaggaga agggcattgc atgaagagtc aacacgctgg aaccgagtgg 7381 gtattctttc agagctgtgc aaggcagtag aatcaaggta tgaaaccgta ggaacttcca 7441 tcatagttat ggccatgact actctagcta gcagtgttaa atcattcagc tacctgagag 7501 gggcccctat aactctctac ggctaacctg aatggactac gacatagtct agtccgccaa 7561 gtctagaaca agtttgtaca aaaaagcagg ctgccaccat ggtgagcaag ggcgaggagc 7621 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt 7681 tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc ctgaagttca 7741 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg 7801 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg 7861 ccatgcccga aggctacgtc caggagcgca ccatcttctt caaggacgac ggcaactaca 7921 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg 7981 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aactacaaca 8041 gccacaacgt ctatatcatg gccgacaagc agaagaacgg catcaaggtg aacttcaaga 8101 tccgccacaa catcgaggac ggcagcgtgc agctcgccga ccactaccag cagaacaccc 8161 ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgagcacc cagtccgccc 8221 tgagcaaaga ccccaacgag aagcgcgatc acatggtcct gctggagttc gtgaccgccg 8281 ccgggatcac tctcggcatg gacgagctgt acaagaagct tagccatggc ttcccgccgg 8341 aggtggagga gcaggatgat ggcacgctgc ccatgtcttg tgcccaggag agcgggatgg 8401 accgtcaccc tgcagcctgt gcttctgcta ggatcaatgt gtaaacccag ctttcttgta 8461 caaagtggtg gcgcgcctat gttacgtgca aaggtgattg tcaccccccg aaagaccata 8521 ttgtgacaca ccctcagtat cacgcccaaa catttacagc cgcggtgtca aaaaccgcgt 8581 ggacgtggtt aacatccctg ctgggaggat cagccgtaat tattataatt ggcttggtgc 8641 tggctactat tgtggccatg tacgtgctga ccaaccagaa acataattga atacagcagc 8701 aattggcaag ctgcttacat agaactcgcg gcgattggca tgccgcctta aaatttttat 8761 tttatttttc ttttcttttc cgaatcggat tttgttttta atatttcaaa aaaaaaaaaa 8821 aaaaaaaaaa aaaaaaaaaa aaaaaaatag ggataacagg gtaattgagc aaaaggccag 8881 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc 8941 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta 9001 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg 9061 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc 9121 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac 9181 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac 9241 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg 9301 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga 9361 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt 9421 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag 9481 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct 9541 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg 9601 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat 9661 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc 9721 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg 9781 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct 9841 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca 9901 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg 9961 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg 10021 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc 10081 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag 10141 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg 10201 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag 10261 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat 10321 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg 10381 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca 10441 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca 10501 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat 10561 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag 10621 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgttagg 10681 gataacaggg taattaatac gactcactat ag //