LOCUS Exported 6166 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Promoter: ppTEF1, minimal Promoter: AOX1. Figure 4. Expresses GFP.. ACCESSION . VERSION . KEYWORDS PP190 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6166) AUTHORS Perez-Pinera P, Han N, Cleto S, Cao J, Purcell O, Shah KA, Lee K, Ram R, Lu TK TITLE Synthetic biology and microbioreactor platforms for programmable production of biologics at the point-of-care. JOURNAL Nat Commun. 2016 Jul 29;7:12211. doi: 10.1038/ncomms12211. PUBMED 27470089 REFERENCE 2 (bases 1 to 6166) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6166 /organism="synthetic DNA construct" /mol_type="other DNA" promoter 157..568 /gene="S. cerevisiae TEF1" /label=TEF1 promoter /note="promoter for EF-1-alpha" promoter 576..623 /label=EM7 promoter /note="synthetic bacterial promoter " CDS 642..1016 /codon_start=1 /gene="Sh ble from Streptoalloteichus hindustanus" /product="antibiotic-binding protein" /label=BleoR /note="confers resistance to bleomycin, phleomycin, and Zeocin(TM)" /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR EFALRDPAGNCVHFVAEEQD" terminator 1082..1329 /gene="S. cerevisiae CYC1" /label=CYC1 terminator /note="transcription terminator for CYC1" primer_bind complement(1098..1116) /label=CYC1 /note="CYC1 transcription termination signal, reverse primer" rep_origin complement(1404..1992) /direction=LEFT /label=ori /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of replication" primer_bind complement(1484..1503) /label=pBR322ori-F /note="pBR322 origin, forward primer" CDS 2947..2967 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4186..4335 /codon_start=1 /product="tetrameric repeat of the minimal activation domain of herpes simplex virus VP16 (Beerli et al., 1998)" /label=VP64 /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF DLDML" CDS 4342..4368 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" terminator 4410..4656 /gene="Pichia pastoris AOX1" /label=AOX1 terminator /note="transcription terminator for AOX1" CDS 5235..5951 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(5280..5301) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(5541..5560) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 5888..5909 /label=EGFP-C /note="EGFP, forward primer" CDS 5976..6005 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 6021..6038 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" terminator 6062..142 /gene="Pichia pastoris AOX1" /label=AOX1 terminator /note="transcription terminator for AOX1" ORIGIN 1 tcttctcgta cgagcttgct cctgatcagc ctatctcgca gctgatgaat atcttgtggt 61 aggggtttgg gaaaatcatt cgagtttgat gtttttcttg gtatttccca ctcctcttca 121 gagtacagaa gattaagtga gaccttcgtt tgtgcggatc ccccacacac catagcttca 181 aaatgtttct actccttttt tactcttcca gattttctcg gactccgcgc atcgccgtac 241 cacttcaaaa cacccaagca cagcatacta aattttccct ctttcttcct ctagggtgtc 301 gttaattacc cgtactaaag gtttggaaaa gaaaaaagag accgcctcgt ttctttttct 361 tcgtcgaaaa aggcaataaa aatttttatc acgtttcttt ttcttgaaat tttttttttt 421 agtttttttc tctttcagtg acctccattg atatttaagt taataaacgg tcttcaattt 481 ctcaagtttc agtttcattt ttcttgttct attacaactt tttttacttc ttgttcatta 541 gaaagaaagc atagcaatct aatctaaggg gcggtgttga caattaatca tcggcatagt 601 atatcggcat agtataatac gacaaggtga ggaactaaac catggccaag ttgaccagtg 661 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc 721 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga 781 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt 841 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc 901 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg 961 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg 1021 tccgacggcg gcccacgggt cccaggcctc ggagatccgt cccccttttc ctttgtcgat 1081 atcatgtaat tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc 1141 gaaaaggaag gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg 1201 ttagtattaa gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg 1261 tacgcatgta acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct 1321 ttaatttgca agctggagac caacatgtga gcaaaaggcc agcaaaaggc caggaaccgt 1381 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa 1441 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt 1501 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg 1561 tccgcctttc tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc 1621 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc 1681 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta 1741 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct 1801 acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc 1861 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa 1921 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa 1981 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa 2041 aactcacgtt aagggatttt ggtcatgaga tcagatctga gctcgtttgg ccgtggccgt 2101 gctcgtcctc gtcggccggc ttgtcgacga cggcggtctc cgtcgtcagg atcatccggg 2161 ccacaagctt gctgacagaa gcctcaagaa aaaaaaaatt cttcttcgac tatgctggag 2221 gcagagatga tcgagccggt agttaactat atatagctaa attggttcca tcacctcgag 2281 tcagcatctg gttacgtaac tctggcaacc agtaacacgc ttaaggtttg gaacaacact 2341 aaactacctt gcggtactac cattgacact acacatcctt aattccaatc ctgtctggcc 2401 tccttcacct tttaaccatc ttgcccattc caactcgtgt cagattgcgt atcaagtgaa 2461 aaaaaaaaaa ttttaaaatc tttaacccaa tcaggtaata actgtcgcct cttttatctg 2521 ccgcactgca tgaggtgtcc ccttagtggg aaagagtact gagccaaccc tggaggacag 2581 caagggaaaa atacctacaa cttgcttcat aatggtcgta aaaacaatcc ttgtcggata 2641 taagtgttgt agactgtccc ttatcctctg cgatgttctt cctctcaaag tttgcgattt 2701 ctctctatca gaattgccat caagagactc aggactaatt tcgcagtccc acacgcactc 2761 gtacatgatt ggctgaaatt tccctaaaga atttcttttt cacgaaaatt tttttttaca 2821 caagattttc agcagatata aaatggagag caggacctcc gctgtgactc ttcttttttt 2881 tcttttattc tcactacata cattttagtt attcgccaac ggcaactagt aaaaatgcgc 2941 ggagctccta agaaaaagcg caaagtccgg ccggcatcta gacccgggga gcgccccttc 3001 cagtgtcgca tttgcatgcg gaacttttcg cgccaggaca ggcttgacag gcatacccgt 3061 actcataccg gtgaaaaacc gtttcagtgt cggatctgta tgcgaaattt ctcccagaag 3121 gagcacttgg cggggcatct acgtacgcac accggcgaga agccattcca atgccgaata 3181 tgcatgcgca acttcagtcg ccgcgacaac ctgaaccggc acctaaaaac ccacctgagg 3241 aacatatgcg gcggaggcac acctgcagct gcgtcgactc tagaggatcc atctgctgga 3301 gacatgagag ctgccaacct ttggccaagc ccgctcatga tcaaacgctc taagaagaac 3361 agcctggcct tgtccctgac ggccgaccag atggtcagtg ccttgttgga tgctgagccc 3421 cccatactct attccgagta tgatcctacc agacccttca gtgaagcttc gatgatgggc 3481 ttactgacca acctggcaga cagggagctg gttcacatga tcaactgggc gaagagggtg 3541 ccaggctttg tggatttgac cctccatgat caggtccacc ttctagaatg tgcctggcta 3601 gagatcctga tgattggtct cgtctggcgc tccatggagc acccagtgaa gctactgttt 3661 gctcctaact tgctcttgga caggaaccag ggaaaatgtg tagagggcat ggtggagatc 3721 ttcgacatgc tgctggctac atcatctcgg ttccgcatga tgaatctgca gggagaggag 3781 tttgtgtgcc tcaaatctat tattttgctt aattctggag tgtacacatt tctgtccagc 3841 accctgaagt ctctggaaga gaaggaccat atccaccgag tcctggacaa gatcacagac 3901 actttgatcc acctgatggc caaggcaggc ctgaccctgc agcagcagca ccagcggctg 3961 gcccagctcc tcctcatcct ctcccacatc aggcacatga gtaacaaagg catggagcat 4021 ctgtacagca tgaagtgcaa gaacgtggtg cccctctatg acctgctgct ggagatgctg 4081 gacgcccacc gcctacatgc gcccactagc cgtggagggg catccgtgga ggagacggac 4141 caaagccact tggccactgc gggctctact tcatcgccta gggccgacgc gctggacgat 4201 ttcgatctcg acatgctggg ttctgatgcc ctcgatgact ttgacctgga tatgttggga 4261 agcgacgcat tggatgactt tgatctggac atgctcggct ccgatgctct ggacgatttc 4321 gatctcgata tgttaattaa ctacccgtac gacgttccgg actacgcttc ttgaggtacc 4381 atcggtagac cggtcttgct agattctaat caagaggatg tcagaatgcc atttgcctga 4441 gagatgcagg cttcattttt gatacttttt tatttgtaac ctatatagta taggattttt 4501 tttgtcattt tgtttcttct cgtacgagct tgctcctgat cagcctatct cgcagctgat 4561 gaatatcttg tggtaggggt ttgggaaaat cattcgagtt tgatgttttt cttggtattt 4621 cccactcctc ttcagagtac agaagattaa gtgagagcta gcatatcata tagaagtcat 4681 cgcggcagat caattcatca atcggagtga ggatattgcg agtttaccac catcaatcgg 4741 agtgaggatc tccaattggt gacggtccag tcatcaatcg gagtgaggat tcagctgctt 4801 ctcgaggccg cacatcaatc ggagtgagga tgtacagggt gggctgttcc accatcaatc 4861 ggagtgagga tttgcgtcaa tggggcggag ttcatcaatc ggagtgagga tatcgaagtc 4921 atcgagagca ctcatcaatc ggagtgagga tatactccac ccattgacgt cacatcaatc 4981 ggagtgagga ttggaaccag aagggtctct tcatcaatcg gagtgaggat accctatggg 5041 cgcgcctaac ccctacttga cagcaatata taaacagaag gaagctgccc tgtcttaaac 5101 cttttttttt atcatcatta ttagcttact ttcataattg cgactggttc caattgacaa 5161 gcttttgatt ttaacgactt ttaacgacaa cttgagaaga tcaaaaaaca actaattatt 5221 cgaaacgcga attcatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc 5281 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg 5341 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg 5401 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc 5461 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg 5521 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg 5581 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca 5641 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg 5701 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca 5761 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc 5821 tgcccgacaa ccactacctg agcacccaga gcgccctgag caaagacccc aacgagaagc 5881 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg 5941 agctgtacaa ggccgcggcg gccgccagct ttctagaaca aaaactcatc tcagaagagg 6001 atctgaatag cgccgtcgac catcatcatc atcatcattg accggtcttg ctagattcta 6061 atcaagagga tgtcagaatg ccatttgcct gagagatgca ggcttcattt ttgatacttt 6121 tttatttgta acctatatag tataggattt tttttgtcat tttgtt //