LOCUS Exported 10580 bp ds-DNA circular SYN 12-MAY-2021 DEFINITION TC-83 I-SceI T7 5'UTR (A3G) P1234 (nsP2 Q739L) SGP XbaI attB1 Kozak mKate opal attB2 AscI (truncated E1) 3'UTR Poly A I-SceI. ACCESSION . VERSION . KEYWORDS pTK194 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 10580) 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 10580) AUTHORS . TITLE Direct Submission JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..10580 /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..8312 /codon_start=1 /product="monomeric derivative of red fluorescent protein from Entacmaea quadricolor (Merzlyak et al., 2007)" /label=TagRFP /note="mammalian codon-optimized" /translation="MVSKGEELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTM RIKVVEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG VLTATQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTEMLYPADGGLEGRSD MALKLVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAV ARYCDLPSKLGHKLN" protein_bind complement(8313..8337) /gene="mutant version of attB" /label=attB2 /bound_moiety="BP Clonase(TM)" /note="recombination site for the Gateway(R) BP reaction" rep_origin complement(8790..9378) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(8870..8889) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS complement(9549..10409) /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 10172..10191 /label=Amp-R /note="Ampicillin resistance gene, reverse primer" promoter complement(10410..10514) /gene="bla" /label=AmpR promoter promoter 10563..10580 /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 ggtgtctaag ggcgaagagc 7621 tgattaagga gaacatgcac atgaagctgt acatggaggg caccgtgaac aaccaccact 7681 tcaagtgcac atccgagggc gaaggcaagc cctacgaggg cacccagacc atgagaatca 7741 aggtggtcga gggcggccct ctccccttcg ccttcgacat cctggctacc agcttcatgt 7801 acggcagcaa aaccttcatc aaccacaccc agggcatccc cgacttcttt aagcagtcct 7861 tccctgaggg cttcacatgg gagagagtca ccacatacga agacgggggc gtgctgaccg 7921 ctacccagga caccagcctc caggacggct gcctcatcta caacgtcaag atcagagggg 7981 tgaacttccc atccaacggc cctgtgatgc agaagaaaac actcggctgg gaggcctcca 8041 ccgagatgct gtaccccgct gacggcggcc tggaaggcag aagcgacatg gccctgaagc 8101 tcgtgggcgg gggccacctg atctgcaact tgaagaccac atacagatcc aagaaacccg 8161 ctaagaacct caagatgccc ggcgtctact atgtggacag aagactggaa agaatcaagg 8221 aggccgacaa agagacctac gtcgagcagc acgaggtggc tgtggccaga tactgcgacc 8281 tccctagcaa actggggcac aaacttaatt gaacccagct ttcttgtaca aagtggtggc 8341 gcgcctatgt tacgtgcaaa ggtgattgtc accccccgaa agaccatatt gtgacacacc 8401 ctcagtatca cgcccaaaca tttacagccg cggtgtcaaa aaccgcgtgg acgtggttaa 8461 catccctgct gggaggatca gccgtaatta ttataattgg cttggtgctg gctactattg 8521 tggccatgta cgtgctgacc aaccagaaac ataattgaat acagcagcaa ttggcaagct 8581 gcttacatag aactcgcggc gattggcatg ccgccttaaa atttttattt tatttttctt 8641 ttcttttccg aatcggattt tgtttttaat atttcaaaaa aaaaaaaaaa aaaaaaaaaa 8701 aaaaaaaaaa aaaaataggg ataacagggt aattgagcaa aaggccagca aaaggccagg 8761 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 8821 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 8881 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 8941 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 9001 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 9061 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 9121 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 9181 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt 9241 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 9301 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 9361 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 9421 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 9481 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 9541 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 9601 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 9661 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 9721 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 9781 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 9841 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 9901 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 9961 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 10021 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 10081 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 10141 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 10201 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 10261 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 10321 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 10381 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 10441 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 10501 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgttaggga taacagggta 10561 attaatacga ctcactatag //