LOCUS       sequence-147734-        4309 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Supplemental Figure 4. Promoter WT GAP. Expresses GFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4309)
  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 4309)
  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: PP149
FEATURES             Location/Qualifiers
     source          1..4309
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      412..659
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(428..446)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(734..1322)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(814..833)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1442..1491
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     promoter        2216..2692
                     /label=GAP promoter
                     /note="promoter for Pichia pastoris 
                     glyceraldehyde-3-phosphate dehydrogenase"
     CDS             join(2708..2710,2711..2713,2714..3424)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2708..2710,2711..2713,2714..3424)
                     /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(2753..2774)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3014..3033)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3361..3382
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3449..3478
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3494..3511
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3535..3781
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3796..4207
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        4215..4262
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             join(4281..4309,1..346)
                     /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"
ORIGIN
        1 gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc tcgggttctc
       61 ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga ccctgttcat
      121 cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg
      181 cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc
      241 cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg ccctgcgcga
      301 cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg tccgacggcg
      361 gcccacgggt cccaggcctc ggagatccgt cccccttttc ctttgtcgat atcatgtaat
      421 tagttatgtc acgcttacat tcacgccctc cccccacatc cgctctaacc gaaaaggaag
      481 gagttagaca acctgaagtc taggtcccta tttatttttt tatagttatg ttagtattaa
      541 gaacgttatt tatatttcaa atttttcttt tttttctgta cagacgcgtg tacgcatgta
      601 acattatact gaaaaccttg cttgagaagg ttttgggacg ctcgaaggct ttaatttgca
      661 agctggagac caacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
      721 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
      781 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
      841 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
      901 tcccttcggg aagcgtggcg ctttctcaat gctcacgctg taggtatctc agttcggtgt
      961 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     1021 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     1081 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     1141 tgaagtggtg gcctaactac ggctacacta gaaggacagt atttggtatc tgcgctctgc
     1201 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     1261 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     1321 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     1381 aagggatttt ggtcatgaga tcagatctga gctcgtttgg ccgtggccgt gctcgtcctc
     1441 gtcggccggc ttgtcgacga cggcggtctc cgtcgtcagg atcatccggg ccacaagctt
     1501 gctgacagaa gcctcaagaa aaaaaaaatt cttcttcgac tatgctggag gcagagatga
     1561 tcgagccggt agttaactat atatagctaa attggttcca tcacctcgag tcaattcttg
     1621 atttagtata cacataacca aatttggatc aagtttgaag taaaacttta acttcagctc
     1681 cttacatttg cactaagatc tctgctactc tggtcccaag tgaaccacct tttggaccct
     1741 attgaccgga ccttaacttg ccaaacctaa acgcttaatg cctcagacgt tttaatgcct
     1801 ctcaacacct ccaaggttgc tttcttgagc atgcctacta gggactttaa cgaactgtgg
     1861 ggttgcagac agtttcaggc gtgtcccgac caatatggcc tactagactc tctgaaaaat
     1921 cacagttttc cagtagttcc gatcaaatta ccatcgaaat ggtcccataa acggacattt
     1981 gacatccgtt cctgaattat agtcttccac cgtggatcat ggtgttcctt tttttcccaa
     2041 agaatatcag catcccttaa ctacgttagg tcagtgatga caatggacca aattgttgca
     2101 aggtttttct ttttctttca tcggcacatt tcagcctcac atgcgactat tatcgatcaa
     2161 tgaaatccat caagattgaa atcttaaaat tgcccctttc acttgacagg atcctttttt
     2221 gtagaaatgt cttggtgtcc tcgtccaatc aggtagccat ctctgaaata tctggctccg
     2281 ttgcaactcc gaacgacctg ctggcaacgt aaaattctcc ggggtaaaac tttaatgtgg
     2341 agtaatggaa ccagaaacgt ctcttccctt ctctctcctt ccaccgcccg ttaccgtccc
     2401 taggaaattt tactctgctg gagagcttct tctacggccc ccttgcagca atgctcttcc
     2461 cagcattacg ttgcgggtaa aacggaggtc gtgtacccga cctagcagcc cagggatgga
     2521 aaagtcccgg ccgtcgctgg caataatagc gggcggacgc atgtcatgag attattggaa
     2581 accaccagaa tcgaatataa aaggcgaaca cctttcccaa ttttggtttc ccctgaccca
     2641 aagactttaa atttaattta tttgtcccta tttcaatcaa ttgaacaact atcaaaacac
     2701 agaattcatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca tcctggtcga
     2761 gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc
     2821 cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg
     2881 gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct accccgacca
     2941 catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac
     3001 catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga
     3061 caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg gcaacatcct
     3121 ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg ccgacaagca
     3181 gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg gcagcgtgca
     3241 gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc tgctgcccga
     3301 caaccactac ctgagcaccc agagcgccct gagcaaagac cccaacgaga agcgcgatca
     3361 catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg acgagctgta
     3421 caaggccgcg gcggccgcca gctttctaga acaaaaactc atctcagaag aggatctgaa
     3481 tagcgccgtc gaccatcatc atcatcatca ttgaccggtc ttgctagatt ctaatcaaga
     3541 ggatgtcaga atgccatttg cctgagagat gcaggcttca tttttgatac ttttttattt
     3601 gtaacctata tagtatagga ttttttttgt cattttgttt cttctcgtac gagcttgctc
     3661 ctgatcagcc tatctcgcag ctgatgaata tcttgtggta ggggtttggg aaaatcattc
     3721 gagtttgatg tttttcttgg tatttcccac tcctcttcag agtacagaag attaagtgag
     3781 accttcgttt gtgcggatcc cccacacacc atagcttcaa aatgtttcta ctcctttttt
     3841 actcttccag attttctcgg actccgcgca tcgccgtacc acttcaaaac acccaagcac
     3901 agcatactaa attttccctc tttcttcctc tagggtgtcg ttaattaccc gtactaaagg
     3961 tttggaaaag aaaaaagaga ccgcctcgtt tctttttctt cgtcgaaaaa ggcaataaaa
     4021 atttttatca cgtttctttt tcttgaaatt ttttttttta gtttttttct ctttcagtga
     4081 cctccattga tatttaagtt aataaacggt cttcaatttc tcaagtttca gtttcatttt
     4141 tcttgttcta ttacaacttt ttttacttct tgttcattag aaagaaagca tagcaatcta
     4201 atctaagggg cggtgttgac aattaatcat cggcatagta tatcggcata gtataatacg
     4261 acaaggtgag gaactaaacc atggccaagt tgaccagtgc cgttccggt
//
