LOCUS       sequence-147707-        6649 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promoter: GAP6, minimal Promoter: mCYC. Figure 4. Expresses GFP..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6649)
  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 6649)
  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: PP258
FEATURES             Location/Qualifiers
     source          1..6649
                     /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)"
     protein_bind    3324..3342
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    3345..3363
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             3433..3453
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4672..4821
                     /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             4828..4854
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      4896..5142
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(5718..5720,5721..5723,5724..6434)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(5718..5720,5721..5723,5724..6434)
                     /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(5763..5784)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(6024..6043)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6371..6392
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             6459..6488
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             6504..6521
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      join(6545..6649,1..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 tcctgaccca aagactttaa atttccctat cagtgataga gatctcccta tcagtgatag
     3361 agataattta tttgtcccta tttcaatcaa ttgaacaact atcaaaacac actagtaaaa
     3421 atgcgcggag ctcctaagaa aaagcgcaaa gtccggccgg catctagacc cggggagcgc
     3481 cccttccagt gtcgcatttg catgcggaac ttttcgcgcc aggacaggct tgacaggcat
     3541 acccgtactc ataccggtga aaaaccgttt cagtgtcgga tctgtatgcg aaatttctcc
     3601 cagaaggagc acttggcggg gcatctacgt acgcacaccg gcgagaagcc attccaatgc
     3661 cgaatatgca tgcgcaactt cagtcgccgc gacaacctga accggcacct aaaaacccac
     3721 ctgaggaaca tatgcggcgg aggcacacct gcagctgcgt cgactctaga ggatccatct
     3781 gctggagaca tgagagctgc caacctttgg ccaagcccgc tcatgatcaa acgctctaag
     3841 aagaacagcc tggccttgtc cctgacggcc gaccagatgg tcagtgcctt gttggatgct
     3901 gagcccccca tactctattc cgagtatgat cctaccagac ccttcagtga agcttcgatg
     3961 atgggcttac tgaccaacct ggcagacagg gagctggttc acatgatcaa ctgggcgaag
     4021 agggtgccag gctttgtgga tttgaccctc catgatcagg tccaccttct agaatgtgcc
     4081 tggctagaga tcctgatgat tggtctcgtc tggcgctcca tggagcaccc agtgaagcta
     4141 ctgtttgctc ctaacttgct cttggacagg aaccagggaa aatgtgtaga gggcatggtg
     4201 gagatcttcg acatgctgct ggctacatca tctcggttcc gcatgatgaa tctgcaggga
     4261 gaggagtttg tgtgcctcaa atctattatt ttgcttaatt ctggagtgta cacatttctg
     4321 tccagcaccc tgaagtctct ggaagagaag gaccatatcc accgagtcct ggacaagatc
     4381 acagacactt tgatccacct gatggccaag gcaggcctga ccctgcagca gcagcaccag
     4441 cggctggccc agctcctcct catcctctcc cacatcaggc acatgagtaa caaaggcatg
     4501 gagcatctgt acagcatgaa gtgcaagaac gtggtgcccc tctatgacct gctgctggag
     4561 atgctggacg cccaccgcct acatgcgccc actagccgtg gaggggcatc cgtggaggag
     4621 acggaccaaa gccacttggc cactgcgggc tctacttcat cgcctagggc cgacgcgctg
     4681 gacgatttcg atctcgacat gctgggttct gatgccctcg atgactttga cctggatatg
     4741 ttgggaagcg acgcattgga tgactttgat ctggacatgc tcggctccga tgctctggac
     4801 gatttcgatc tcgatatgtt aattaactac ccgtacgacg ttccggacta cgcttcttga
     4861 ggtaccatcg gtagaccggt cttgctagat tctaatcaag aggatgtcag aatgccattt
     4921 gcctgagaga tgcaggcttc atttttgata cttttttatt tgtaacctat atagtatagg
     4981 attttttttg tcattttgtt tcttctcgta cgagcttgct cctgatcagc ctatctcgca
     5041 gctgatgaat atcttgtggt aggggtttgg gaaaatcatt cgagtttgat gtttttcttg
     5101 gtatttccca ctcctcttca gagtacagaa gattaagtga gagctagcat atcatataga
     5161 agtcatcgcg gcagatcaat tcatcaatcg gagtgaggat attgcgagtt taccaccatc
     5221 aatcggagtg aggatctcca attggtgacg gtccagtcat caatcggagt gaggattcag
     5281 ctgcttctcg aggccgcaca tcaatcggag tgaggatgta cagggtgggc tgttccacca
     5341 tcaatcggag tgaggatttg cgtcaatggg gcggagttca tcaatcggag tgaggatatc
     5401 gaagtcatcg agagcactca tcaatcggag tgaggatata ctccacccat tgacgtcaca
     5461 tcaatcggag tgaggattgg aaccagaagg gtctcttcat caatcggagt gaggataccc
     5521 tatgggcgcg ccaccctatg gcatgcatgt gctctgtatg tatataaaac tcttgttttc
     5581 ttcttttctc taaatattct ttccttatac attaggtcct ttgtagcata aattactata
     5641 cttctataga cacgcaaaca caaatacaca cactaaatta ccggatcaat tcgaagctta
     5701 aatcgaaacg cgaattcatg gtgagcaagg gcgaggagct gttcaccggg gtggtgccca
     5761 tcctggtcga gctggacggc gacgtaaacg gccacaagtt cagcgtgtcc ggcgagggcg
     5821 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     5881 ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg cgtgcagtgc ttcagccgct
     5941 accccgacca catgaagcag cacgacttct tcaagtccgc catgcccgaa ggctacgtcc
     6001 aggagcgcac catcttcttc aaggacgacg gcaactacaa gacccgcgcc gaggtgaagt
     6061 tcgagggcga caccctggtg aaccgcatcg agctgaaggg catcgacttc aaggaggacg
     6121 gcaacatcct ggggcacaag ctggagtaca actacaacag ccacaacgtc tatatcatgg
     6181 ccgacaagca gaagaacggc atcaaggtga acttcaagat ccgccacaac atcgaggacg
     6241 gcagcgtgca gctcgccgac cactaccagc agaacacccc catcggcgac ggccccgtgc
     6301 tgctgcccga caaccactac ctgagcaccc agagcgccct gagcaaagac cccaacgaga
     6361 agcgcgatca catggtcctg ctggagttcg tgaccgccgc cgggatcact ctcggcatgg
     6421 acgagctgta caaggccgcg gcggccgcca gctttctaga acaaaaactc atctcagaag
     6481 aggatctgaa tagcgccgtc gaccatcatc atcatcatca ttgaccggtc ttgctagatt
     6541 ctaatcaaga ggatgtcaga atgccatttg cctgagagat gcaggcttca tttttgatac
     6601 ttttttattt gtaacctata tagtatagga ttttttttgt cattttgtt
//
