LOCUS       sequence-171563-        6613 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promoter: GAP, minimal Promoter: AOX1. Figure 4. Expresses GFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6613)
  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 6613)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2016-07-29"; volume: "7:12211. doi"; pages: " 
            10.1038/ncomms12211"
COMMENT     Sequence Label: PP245
FEATURES             Location/Qualifiers
     source          1..6613
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(48..636)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(128..147)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    756..805
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     promoter        1530..2006
                     /label=GAP promoter
                     /note="promoter for Pichia pastoris 
                     glyceraldehyde-3-phosphate dehydrogenase"
     CDS             2037..2057
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3276..3425
                     /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             3432..3458
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      3500..3746
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(4325..4327,4328..4330,4331..5041)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4325..4327,4328..4330,4331..5041)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4370..4391)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4631..4650)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4978..4999
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5066..5095
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5111..5128
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      5152..5398
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        5413..5826
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        5833..5880
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5899..6273
                     /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      6339..6586
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(6355..6373)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
ORIGIN
        1 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
       61 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      121 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
      181 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
      241 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
      301 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
      361 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
      421 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
      481 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
      541 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
      601 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
      661 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagatcagat
      721 ctgagctcgt ttggccgtgg ccgtgctcgt cctcgtcggc cggcttgtcg acgacggcgg
      781 tctccgtcgt caggatcatc cgggccacaa gcttgctgac agaagcctca agaaaaaaaa
      841 aattcttctt cgactatgct ggaggcagag atgatcgagc cggtagttaa ctatatatag
      901 ctaaattggt tccatcacct cgagtcaatt cttgatttag tatacacata accaaatttg
      961 gatcaagttt gaagtaaaac tttaacttca gctccttaca tttgcactaa gatctctgct
     1021 actctggtcc caagtgaacc accttttgga ccctattgac cggaccttaa cttgccaaac
     1081 ctaaacgctt aatgcctcag acgttttaat gcctctcaac acctccaagg ttgctttctt
     1141 gagcatgcct actagggact ttaacgaact gtggggttgc agacagtttc aggcgtgtcc
     1201 cgaccaatat ggcctactag actctctgaa aaatcacagt tttccagtag ttccgatcaa
     1261 attaccatcg aaatggtccc ataaacggac atttgacatc cgttcctgaa ttatagtctt
     1321 ccaccgtgga tcatggtgtt cctttttttc ccaaagaata tcagcatccc ttaactacgt
     1381 taggtcagtg atgacaatgg accaaattgt tgcaaggttt ttctttttct ttcatcggca
     1441 catttcagcc tcacatgcga ctattatcga tcaatgaaat ccatcaagat tgaaatctta
     1501 aaattgcccc tttcacttga caggatcctt ttttgtagaa atgtcttggt gtcctcgtcc
     1561 aatcaggtag ccatctctga aatatctggc tccgttgcaa ctccgaacga cctgctggca
     1621 acgtaaaatt ctccggggta aaactttaat gtggagtaat ggaaccagaa acgtctcttc
     1681 ccttctctct ccttccaccg cccgttaccg tccctaggaa attttactct gctggagagc
     1741 ttcttctacg gcccccttgc agcaatgctc ttcccagcat tacgttgcgg gtaaaacgga
     1801 ggtcgtgtac ccgacctagc agcccaggga tggaaaagtc ccggccgtcg ctggcaataa
     1861 tagcgggcgg acgcatgtca tgagattatt ggaaaccacc agaatcgaat ataaaaggcg
     1921 aacacctttc ccaattttgg tttcccctga cccaaagact ttaaatttaa tttatttgtc
     1981 cctatttcaa tcaattgaac aactatcaaa acacactagt aaaaatgcgc ggagctccta
     2041 agaaaaagcg caaagtccgg ccggcatcta gacccgggga gcgccccttc cagtgtcgca
     2101 tttgcatgcg gaacttttcg cgccaggaca ggcttgacag gcatacccgt actcataccg
     2161 gtgaaaaacc gtttcagtgt cggatctgta tgcgaaattt ctcccagaag gagcacttgg
     2221 cggggcatct acgtacgcac accggcgaga agccattcca atgccgaata tgcatgcgca
     2281 acttcagtcg ccgcgacaac ctgaaccggc acctaaaaac ccacctgagg aacatatgcg
     2341 gcggaggcac acctgcagct gcgtcgactc tagaggatcc atctgctgga gacatgagag
     2401 ctgccaacct ttggccaagc ccgctcatga tcaaacgctc taagaagaac agcctggcct
     2461 tgtccctgac ggccgaccag atggtcagtg ccttgttgga tgctgagccc cccatactct
     2521 attccgagta tgatcctacc agacccttca gtgaagcttc gatgatgggc ttactgacca
     2581 acctggcaga cagggagctg gttcacatga tcaactgggc gaagagggtg ccaggctttg
     2641 tggatttgac cctccatgat caggtccacc ttctagaatg tgcctggcta gagatcctga
     2701 tgattggtct cgtctggcgc tccatggagc acccagtgaa gctactgttt gctcctaact
     2761 tgctcttgga caggaaccag ggaaaatgtg tagagggcat ggtggagatc ttcgacatgc
     2821 tgctggctac atcatctcgg ttccgcatga tgaatctgca gggagaggag tttgtgtgcc
     2881 tcaaatctat tattttgctt aattctggag tgtacacatt tctgtccagc accctgaagt
     2941 ctctggaaga gaaggaccat atccaccgag tcctggacaa gatcacagac actttgatcc
     3001 acctgatggc caaggcaggc ctgaccctgc agcagcagca ccagcggctg gcccagctcc
     3061 tcctcatcct ctcccacatc aggcacatga gtaacaaagg catggagcat ctgtacagca
     3121 tgaagtgcaa gaacgtggtg cccctctatg acctgctgct ggagatgctg gacgcccacc
     3181 gcctacatgc gcccactagc cgtggagggg catccgtgga ggagacggac caaagccact
     3241 tggccactgc gggctctact tcatcgccta gggccgacgc gctggacgat ttcgatctcg
     3301 acatgctggg ttctgatgcc ctcgatgact ttgacctgga tatgttggga agcgacgcat
     3361 tggatgactt tgatctggac atgctcggct ccgatgctct ggacgatttc gatctcgata
     3421 tgttaattaa ctacccgtac gacgttccgg actacgcttc ttgaggtacc atcggtagac
     3481 cggtcttgct agattctaat caagaggatg tcagaatgcc atttgcctga gagatgcagg
     3541 cttcattttt gatacttttt tatttgtaac ctatatagta taggattttt tttgtcattt
     3601 tgtttcttct cgtacgagct tgctcctgat cagcctatct cgcagctgat gaatatcttg
     3661 tggtaggggt ttgggaaaat cattcgagtt tgatgttttt cttggtattt cccactcctc
     3721 ttcagagtac agaagattaa gtgagagcta gcatatcata tagaagtcat cgcggcagat
     3781 caattcatca atcggagtga ggatattgcg agtttaccac catcaatcgg agtgaggatc
     3841 tccaattggt gacggtccag tcatcaatcg gagtgaggat tcagctgctt ctcgaggccg
     3901 cacatcaatc ggagtgagga tgtacagggt gggctgttcc accatcaatc ggagtgagga
     3961 tttgcgtcaa tggggcggag ttcatcaatc ggagtgagga tatcgaagtc atcgagagca
     4021 ctcatcaatc ggagtgagga tatactccac ccattgacgt cacatcaatc ggagtgagga
     4081 ttggaaccag aagggtctct tcatcaatcg gagtgaggat accctatggg cgcgcctaac
     4141 ccctacttga cagcaatata taaacagaag gaagctgccc tgtcttaaac cttttttttt
     4201 atcatcatta ttagcttact ttcataattg cgactggttc caattgacaa gcttttgatt
     4261 ttaacgactt ttaacgacaa cttgagaaga tcaaaaaaca actaattatt cgaaacgcga
     4321 attcatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     4381 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg gcgatgccac
     4441 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     4501 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     4561 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg agcgcaccat
     4621 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     4681 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     4741 gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg acaagcagaa
     4801 gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca gcgtgcagct
     4861 cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc tgcccgacaa
     4921 ccactacctg agcacccaga gcgccctgag caaagacccc aacgagaagc gcgatcacat
     4981 ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg agctgtacaa
     5041 ggccgcggcg gccgccagct ttctagaaca aaaactcatc tcagaagagg atctgaatag
     5101 cgccgtcgac catcatcatc atcatcattg accggtcttg ctagattcta atcaagagga
     5161 tgtcagaatg ccatttgcct gagagatgca ggcttcattt ttgatacttt tttatttgta
     5221 acctatatag tataggattt tttttgtcat tttgtttctt ctcgtacgag cttgctcctg
     5281 atcagcctat ctcgcagctg atgaatatct tgtggtaggg gtttgggaaa atcattcgag
     5341 tttgatgttt ttcttggtat ttcccactcc tcttcagagt acagaagatt aagtgagacc
     5401 ttcgtttgtg cggatccccc acacaccata gcttcaaaat gtttctactc cttttttact
     5461 cttccagatt ttctcggact ccgcgcatcg ccgtaccact tcaaaacacc caagcacagc
     5521 atactaaatt tcccctcttt cttcctctag ggtgtcgtta attacccgta ctaaaggttt
     5581 ggaaaagaaa aaagagaccg cctcgtttct ttttcttcgt cgaaaaaggc aataaaaatt
     5641 tttatcacgt ttctttttct tgaaaatttt tttttttgat ttttttctct ttcgatgacc
     5701 tcccattgat atttaagtta ataaacggtc ttcaatttct caagtttcag tttcattttt
     5761 cttgttctat tacaactttt tttacttctt gctcattaga aagaaagcat agcaatctaa
     5821 tctaagggcg gtgttgacaa ttaatcatcg gcatagtata tcggcatagt ataatacgac
     5881 aaggtgagga actaaaccat ggccaagttg accagtgccg ttccggtgct caccgcgcgc
     5941 gacgtcgccg gagcggtcga gttctggacc gaccggctcg ggttctcccg ggacttcgtg
     6001 gaggacgact tcgccggtgt ggtccgggac gacgtgaccc tgttcatcag cgcggtccag
     6061 gaccaggtgg tgccggacaa caccctggcc tgggtgtggg tgcgcggcct ggacgagctg
     6121 tacgccgagt ggtcggaggt cgtgtccacg aacttccggg acgcctccgg gccggccatg
     6181 accgagatcg gcgagcagcc gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac
     6241 tgcgtgcact tcgtggccga ggagcaggac tgacacgtcc gacgcggccc gacgggtccg
     6301 aggcctcgga gatccgtccc ccttttcctt tgtcgatatc atgtaattag ttatgtcacg
     6361 cttacattca cgccctcccc ccacatccgc tctaaccgaa aaggaaggag ttagacaacc
     6421 tgaagtctag gtccctattt atttttttat agttatgtta gtattaagaa cgttatttat
     6481 atttcaaatt tttctttttt ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa
     6541 aaccttgctt gagaaggttt tgggacgctc gaaggcttta atttgcaagc tggagaccaa
     6601 catgtgagca aaa
//
