LOCUS       sequence-147730-        4040 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Supplemental Figure 4. Promoter GCW14. Expresses GFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4040)
  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 4040)
  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: PP165
FEATURES             Location/Qualifiers
     source          1..4040
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             59..433
                     /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      499..746
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(515..533)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     rep_origin      complement(821..1409)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(901..920)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    1529..1578
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     CDS             join(2526..2528,2529..2531,2532..3242)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2526..2528,2529..2531,2532..3242)
                     /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(2571..2592)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2832..2851)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3179..3200
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3267..3296
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3312..3329
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      3353..3599
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        3614..4025
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        join(4033..4040,1..40)
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
ORIGIN
        1 ttaatcatcg gcatagtata tcggcatagt ataatacgac aaggtgagga actaaaccat
       61 ggccaagttg accagtgccg ttccggtgct caccgcgcgc gacgtcgccg gagcggtcga
      121 gttctggacc gaccggctcg ggttctcccg ggacttcgtg gaggacgact tcgccggtgt
      181 ggtccgggac gacgtgaccc tgttcatcag cgcggtccag gaccaggtgg tgccggacaa
      241 caccctggcc tgggtgtggg tgcgcggcct ggacgagctg tacgccgagt ggtcggaggt
      301 cgtgtccacg aacttccggg acgcctccgg gccggccatg accgagatcg gcgagcagcc
      361 gtgggggcgg gagttcgccc tgcgcgaccc ggccggcaac tgcgtgcact tcgtggccga
      421 ggagcaggac tgacacgtcc gacggcggcc cacgggtccc aggcctcgga gatccgtccc
      481 ccttttcctt tgtcgatatc atgtaattag ttatgtcacg cttacattca cgccctcccc
      541 ccacatccgc tctaaccgaa aaggaaggag ttagacaacc tgaagtctag gtccctattt
      601 atttttttat agttatgtta gtattaagaa cgttatttat atttcaaatt tttctttttt
      661 ttctgtacag acgcgtgtac gcatgtaaca ttatactgaa aaccttgctt gagaaggttt
      721 tgggacgctc gaaggcttta atttgcaagc tggagaccaa catgtgagca aaaggccagc
      781 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
      841 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
      901 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
      961 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcaatgct
     1021 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     1081 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     1141 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     1201 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     1261 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     1321 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     1381 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     1441 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatca gatctgagct
     1501 cgtttggccg tggccgtgct cgtcctcgtc ggccggcttg tcgacgacgg cggtctccgt
     1561 cgtcaggatc atccgggcca caagcttgct gacagaagcc tcaagaaaaa aaaaattctt
     1621 cttcgactat gctggaggca gagatgatcg agccggtagt taactatata tagctaaatt
     1681 ggttccatca cctcgagcag gtgaacccac ctaactattt ttaactggga tccagtgagc
     1741 tcgctgggtg aaagccaacc atcttttgtt tcggggaacc gtgctcgccc cgtaaagtta
     1801 attttttttt cccgcgcagc tttaatcttt cggcagagaa ggcgttttca tcgtagcgtg
     1861 ggaacagaat aatcagttca tgtgctatac aggcacatgg cagcagtcac tattttgctt
     1921 tttaacctta aagtcgttca tcaatcatta actgaccaat cagatttttt gcatttgcca
     1981 cttatctaaa aatacttttg tatctcgcag atacgttcag tggtttccag gacaacaccc
     2041 aaaaaaaggt atcaatgcca ctaggcagtc ggttttattt ttggtcaccc acgcaaagaa
     2101 gcacccacct cttttaggtt ttaagttgtg ggaacagtaa caccgcctag agcttcagga
     2161 aaaaccagta cctgtgaccg caattcacca tgatgcagaa tgttaattta aacgagtgcc
     2221 aaatcaagat ttcaacagac aaatcaatcg atccatagtt acccattcca gccttttcgt
     2281 cgtcgagcct gcttcattcc tgcctcaggt gcataacttt gcatgaaaag tccagattag
     2341 ggcagatttt gagtttaaaa taggaaatat aaacaaatat accgcgaaaa aggtttgttt
     2401 atagcttttc gcctggtgcc gtacggtata aatacatact ctcctccccc ccctggttct
     2461 ctttttcttt tgttacttac attttaccgt tccgtcactc gcttcactca acaacaaaag
     2521 aattcatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     2581 tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca
     2641 cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     2701 ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca
     2761 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     2821 tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca
     2881 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     2941 ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga
     3001 agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc
     3061 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     3121 accactacct gagcacccag agcgccctga gcaaagaccc caacgagaag cgcgatcaca
     3181 tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca
     3241 aggccgcggc ggccgccagc tttctagaac aaaaactcat ctcagaagag gatctgaata
     3301 gcgccgtcga ccatcatcat catcatcatt gaccggtctt gctagattct aatcaagagg
     3361 atgtcagaat gccatttgcc tgagagatgc aggcttcatt tttgatactt ttttatttgt
     3421 aacctatata gtataggatt ttttttgtca ttttgtttct tctcgtacga gcttgctcct
     3481 gatcagccta tctcgcagct gatgaatatc ttgtggtagg ggtttgggaa aatcattcga
     3541 gtttgatgtt tttcttggta tttcccactc ctcttcagag tacagaagat taagtgagac
     3601 cttcgtttgt gcggatcccc cacacaccat agcttcaaaa tgtttctact ccttttttac
     3661 tcttccagat tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag
     3721 catactaaat tttccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt
     3781 tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat
     3841 ttttatcacg tttctttttc ttgaaatttt tttttttagt ttttttctct ttcagtgacc
     3901 tccattgata tttaagttaa taaacggtct tcaatttctc aagtttcagt ttcatttttc
     3961 ttgttctatt acaacttttt ttacttcttg ttcattagaa agaaagcata gcaatctaat
     4021 ctaaggggcg gtgttgacaa
//
