LOCUS       sequence-147687-        6163 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GFP. Figures 3 and 4. Strain 8. Promoter: ppTEF1, minimal 
            Promoter: mCYC. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6163)
  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 6163)
  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: PP228
FEATURES             Location/Qualifiers
     source          1..6163
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     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"
     protein_bind    2112..2161
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     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             join(5232..5234,5235..5237,5238..5948)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5232..5234,5235..5237,5238..5948)
                     /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(5277..5298)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5538..5557)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5885..5906
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5973..6002
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             6018..6035
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      join(6059..6163,1..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 cgcgccaccc tatggcatgc atgtgctctg tatgtatata aaactcttgt tttcttcttt
     5101 tctctaaata ttctttcctt atacattagg tcctttgtag cataaattac tatacttcta
     5161 tagacacgca aacacaaata cacacactaa attaccggat caattcgaag cttaaatcga
     5221 aacgcgaatt catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     5281 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     5341 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     5401 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     5461 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     5521 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     5581 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     5641 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     5701 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     5761 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     5821 ccgacaacca ctacctgagc acccagagcg ccctgagcaa agaccccaac gagaagcgcg
     5881 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     5941 tgtacaaggc cgcggcggcc gccagctttc tagaacaaaa actcatctca gaagaggatc
     6001 tgaatagcgc cgtcgaccat catcatcatc atcattgacc ggtcttgcta gattctaatc
     6061 aagaggatgt cagaatgcca tttgcctgag agatgcaggc ttcatttttg atactttttt
     6121 atttgtaacc tatatagtat aggatttttt ttgtcatttt gtt
//
