LOCUS Exported 6609 bp ds-DNA circular SYN 13-MAY-2021 DEFINITION Promoter: GAP, minimal Promoter: mCYC. Figure 4. Expresses GFP.. ACCESSION . VERSION . KEYWORDS PP257 SOURCE synthetic DNA construct ORGANISM synthetic DNA construct REFERENCE 1 (bases 1 to 6609) 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 6609) AUTHORS . TITLE Direct Submission JOURNAL Exported May 13, 2021 from SnapGene Server 1.1.58 http://www.snapgene.com FEATURES Location/Qualifiers source 1..6609 /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" promoter 2886..3362 /label=GAP promoter /note="promoter for Pichia pastoris glyceraldehyde-3-phosphate dehydrogenase" CDS 3393..3413 /codon_start=1 /product="nuclear localization signal of SV40 (simian virus 40) large T antigen" /label=SV40 NLS /translation="PKKKRKV" CDS 4632..4781 /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 4788..4814 /codon_start=1 /product="HA (human influenza hemagglutinin) epitope tag" /label=HA /translation="YPYDVPDYA" terminator 4856..5102 /gene="Pichia pastoris AOX1" /label=AOX1 terminator /note="transcription terminator for AOX1" CDS 5678..6394 /codon_start=1 /product="enhanced GFP" /label=EGFP /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL EFVTAAGITLGMDELYK" primer_bind complement(5723..5744) /label=EGFP-N /note="EGFP, reverse primer" primer_bind complement(5984..6003) /label=EXFP-R /note="For distinguishing EGFP variants, reverse primer" primer_bind 6331..6352 /label=EGFP-C /note="EGFP, forward primer" CDS 6419..6448 /codon_start=1 /product="Myc (human c-Myc proto-oncogene) epitope tag" /label=Myc /translation="EQKLISEEDL" CDS 6464..6481 /codon_start=1 /product="6xHis affinity tag" /label=6xHis /translation="HHHHHH" terminator 6505..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 tcaattcttg atttagtata cacataacca aatttggatc aagtttgaag taaaacttta 2341 acttcagctc cttacatttg cactaagatc tctgctactc tggtcccaag tgaaccacct 2401 tttggaccct attgaccgga ccttaacttg ccaaacctaa acgcttaatg cctcagacgt 2461 tttaatgcct ctcaacacct ccaaggttgc tttcttgagc atgcctacta gggactttaa 2521 cgaactgtgg ggttgcagac agtttcaggc gtgtcccgac caatatggcc tactagactc 2581 tctgaaaaat cacagttttc cagtagttcc gatcaaatta ccatcgaaat ggtcccataa 2641 acggacattt gacatccgtt cctgaattat agtcttccac cgtggatcat ggtgttcctt 2701 tttttcccaa agaatatcag catcccttaa ctacgttagg tcagtgatga caatggacca 2761 aattgttgca aggtttttct ttttctttca tcggcacatt tcagcctcac atgcgactat 2821 tatcgatcaa tgaaatccat caagattgaa atcttaaaat tgcccctttc acttgacagg 2881 atcctttttt gtagaaatgt cttggtgtcc tcgtccaatc aggtagccat ctctgaaata 2941 tctggctccg ttgcaactcc gaacgacctg ctggcaacgt aaaattctcc ggggtaaaac 3001 tttaatgtgg agtaatggaa ccagaaacgt ctcttccctt ctctctcctt ccaccgcccg 3061 ttaccgtccc taggaaattt tactctgctg gagagcttct tctacggccc ccttgcagca 3121 atgctcttcc cagcattacg ttgcgggtaa aacggaggtc gtgtacccga cctagcagcc 3181 cagggatgga aaagtcccgg ccgtcgctgg caataatagc gggcggacgc atgtcatgag 3241 attattggaa accaccagaa tcgaatataa aaggcgaaca cctttcccaa ttttggtttc 3301 ccctgaccca aagactttaa atttaattta tttgtcccta tttcaatcaa ttgaacaact 3361 atcaaaacac actagtaaaa atgcgcggag ctcctaagaa aaagcgcaaa gtccggccgg 3421 catctagacc cggggagcgc cccttccagt gtcgcatttg catgcggaac ttttcgcgcc 3481 aggacaggct tgacaggcat acccgtactc ataccggtga aaaaccgttt cagtgtcgga 3541 tctgtatgcg aaatttctcc cagaaggagc acttggcggg gcatctacgt acgcacaccg 3601 gcgagaagcc attccaatgc cgaatatgca tgcgcaactt cagtcgccgc gacaacctga 3661 accggcacct aaaaacccac ctgaggaaca tatgcggcgg aggcacacct gcagctgcgt 3721 cgactctaga ggatccatct gctggagaca tgagagctgc caacctttgg ccaagcccgc 3781 tcatgatcaa acgctctaag aagaacagcc tggccttgtc cctgacggcc gaccagatgg 3841 tcagtgcctt gttggatgct gagcccccca tactctattc cgagtatgat cctaccagac 3901 ccttcagtga agcttcgatg atgggcttac tgaccaacct ggcagacagg gagctggttc 3961 acatgatcaa ctgggcgaag agggtgccag gctttgtgga tttgaccctc catgatcagg 4021 tccaccttct agaatgtgcc tggctagaga tcctgatgat tggtctcgtc tggcgctcca 4081 tggagcaccc agtgaagcta ctgtttgctc ctaacttgct cttggacagg aaccagggaa 4141 aatgtgtaga gggcatggtg gagatcttcg acatgctgct ggctacatca tctcggttcc 4201 gcatgatgaa tctgcaggga gaggagtttg tgtgcctcaa atctattatt ttgcttaatt 4261 ctggagtgta cacatttctg tccagcaccc tgaagtctct ggaagagaag gaccatatcc 4321 accgagtcct ggacaagatc acagacactt tgatccacct gatggccaag gcaggcctga 4381 ccctgcagca gcagcaccag cggctggccc agctcctcct catcctctcc cacatcaggc 4441 acatgagtaa caaaggcatg gagcatctgt acagcatgaa gtgcaagaac gtggtgcccc 4501 tctatgacct gctgctggag atgctggacg cccaccgcct acatgcgccc actagccgtg 4561 gaggggcatc cgtggaggag acggaccaaa gccacttggc cactgcgggc tctacttcat 4621 cgcctagggc cgacgcgctg gacgatttcg atctcgacat gctgggttct gatgccctcg 4681 atgactttga cctggatatg ttgggaagcg acgcattgga tgactttgat ctggacatgc 4741 tcggctccga tgctctggac gatttcgatc tcgatatgtt aattaactac ccgtacgacg 4801 ttccggacta cgcttcttga ggtaccatcg gtagaccggt cttgctagat tctaatcaag 4861 aggatgtcag aatgccattt gcctgagaga tgcaggcttc atttttgata cttttttatt 4921 tgtaacctat atagtatagg attttttttg tcattttgtt tcttctcgta cgagcttgct 4981 cctgatcagc ctatctcgca gctgatgaat atcttgtggt aggggtttgg gaaaatcatt 5041 cgagtttgat gtttttcttg gtatttccca ctcctcttca gagtacagaa gattaagtga 5101 gagctagcat atcatataga agtcatcgcg gcagatcaat tcatcaatcg gagtgaggat 5161 attgcgagtt taccaccatc aatcggagtg aggatctcca attggtgacg gtccagtcat 5221 caatcggagt gaggattcag ctgcttctcg aggccgcaca tcaatcggag tgaggatgta 5281 cagggtgggc tgttccacca tcaatcggag tgaggatttg cgtcaatggg gcggagttca 5341 tcaatcggag tgaggatatc gaagtcatcg agagcactca tcaatcggag tgaggatata 5401 ctccacccat tgacgtcaca tcaatcggag tgaggattgg aaccagaagg gtctcttcat 5461 caatcggagt gaggataccc tatgggcgcg ccaccctatg gcatgcatgt gctctgtatg 5521 tatataaaac tcttgttttc ttcttttctc taaatattct ttccttatac attaggtcct 5581 ttgtagcata aattactata cttctataga cacgcaaaca caaatacaca cactaaatta 5641 ccggatcaat tcgaagctta aatcgaaacg cgaattcatg gtgagcaagg gcgaggagct 5701 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt 5761 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat 5821 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg 5881 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc 5941 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa 6001 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg 6061 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag 6121 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat 6181 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc 6241 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agagcgccct 6301 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc 6361 cgggatcact ctcggcatgg acgagctgta caaggccgcg gcggccgcca gctttctaga 6421 acaaaaactc atctcagaag aggatctgaa tagcgccgtc gaccatcatc atcatcatca 6481 ttgaccggtc ttgctagatt ctaatcaaga ggatgtcaga atgccatttg cctgagagat 6541 gcaggcttca tttttgatac ttttttattt gtaacctata tagtatagga ttttttttgt 6601 cattttgtt //