LOCUS       sequence-147708-        6609 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promoter: GAP, minimal Promoter: mCYC. Figure 4. Expresses GFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
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 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: PP257
FEATURES             Location/Qualifiers
     source          1..6609
                     /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)"
     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             join(5678..5680,5681..5683,5684..6394)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5678..5680,5681..5683,5684..6394)
                     /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(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      join(6505..6609,1..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 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
//
