LOCUS       sequence-147678-        5918 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Promoter: scTEF1, minimal Promoter: GAP. Figure 2 and 4..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5918)
  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 5918)
  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: PP255
FEATURES             Location/Qualifiers
     source          1..5918
                     /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        2282..2688
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             2712..2732
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3951..4100
                     /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             4107..4133
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      4175..4421
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(4987..4989,4990..4992,4993..5703)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4987..4989,4990..4992,4993..5703)
                     /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(5032..5053)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(5293..5312)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5640..5661
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             5728..5757
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             5773..5790
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      join(5814..5918,1..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 tccacacacc atagcttcaa aatgtttcta ctcctttttt actcttccag attttctcgg
     2341 actccgcgca tcgccgtacc acttcaaaac acccaagcac agcatactaa atttcccctc
     2401 tttcttcctc tagggtgtcg ttaattaccc gtactaaagg tttggaaaag aaaaaagaga
     2461 ccgcctcgtt tctttttctt cgtcgaaaaa ggcaataaaa atttttatca cgtttctttt
     2521 tcttgaaaat tttttttttg atttttttct ctttcgatga cctcccattg atatttaagt
     2581 taataaacgg tcttcaattt ctcaagtttc agtttcattt ttcttgttct attacaactt
     2641 tttttacttc ttgctcatta gaaagaaagc atagcaatct aatctaagga ctagtaaaaa
     2701 tgcgcggagc tcctaagaaa aagcgcaaag tccggccggc atctagaccc ggggagcgcc
     2761 ccttccagtg tcgcatttgc atgcggaact tttcgcgcca ggacaggctt gacaggcata
     2821 cccgtactca taccggtgaa aaaccgtttc agtgtcggat ctgtatgcga aatttctccc
     2881 agaaggagca cttggcgggg catctacgta cgcacaccgg cgagaagcca ttccaatgcc
     2941 gaatatgcat gcgcaacttc agtcgccgcg acaacctgaa ccggcaccta aaaacccacc
     3001 tgaggaacat atgcggcgga ggcacacctg cagctgcgtc gactctagag gatccatctg
     3061 ctggagacat gagagctgcc aacctttggc caagcccgct catgatcaaa cgctctaaga
     3121 agaacagcct ggccttgtcc ctgacggccg accagatggt cagtgccttg ttggatgctg
     3181 agccccccat actctattcc gagtatgatc ctaccagacc cttcagtgaa gcttcgatga
     3241 tgggcttact gaccaacctg gcagacaggg agctggttca catgatcaac tgggcgaaga
     3301 gggtgccagg ctttgtggat ttgaccctcc atgatcaggt ccaccttcta gaatgtgcct
     3361 ggctagagat cctgatgatt ggtctcgtct ggcgctccat ggagcaccca gtgaagctac
     3421 tgtttgctcc taacttgctc ttggacagga accagggaaa atgtgtagag ggcatggtgg
     3481 agatcttcga catgctgctg gctacatcat ctcggttccg catgatgaat ctgcagggag
     3541 aggagtttgt gtgcctcaaa tctattattt tgcttaattc tggagtgtac acatttctgt
     3601 ccagcaccct gaagtctctg gaagagaagg accatatcca ccgagtcctg gacaagatca
     3661 cagacacttt gatccacctg atggccaagg caggcctgac cctgcagcag cagcaccagc
     3721 ggctggccca gctcctcctc atcctctccc acatcaggca catgagtaac aaaggcatgg
     3781 agcatctgta cagcatgaag tgcaagaacg tggtgcccct ctatgacctg ctgctggaga
     3841 tgctggacgc ccaccgccta catgcgccca ctagccgtgg aggggcatcc gtggaggaga
     3901 cggaccaaag ccacttggcc actgcgggct ctacttcatc gcctagggcc gacgcgctgg
     3961 acgatttcga tctcgacatg ctgggttctg atgccctcga tgactttgac ctggatatgt
     4021 tgggaagcga cgcattggat gactttgatc tggacatgct cggctccgat gctctggacg
     4081 atttcgatct cgatatgtta attaactacc cgtacgacgt tccggactac gcttcttgag
     4141 gtaccatcgg tagaccggtc ttgctagatt ctaatcaaga ggatgtcaga atgccatttg
     4201 cctgagagat gcaggcttca tttttgatac ttttttattt gtaacctata tagtatagga
     4261 ttttttttgt cattttgttt cttctcgtac gagcttgctc ctgatcagcc tatctcgcag
     4321 ctgatgaata tcttgtggta ggggtttggg aaaatcattc gagtttgatg tttttcttgg
     4381 tatttcccac tcctcttcag agtacagaag attaagtgag agctagcata tcatatagaa
     4441 gtcatcgcgg cagatcaatt catcaatcgg agtgaggata ttgcgagttt accaccatca
     4501 atcggagtga ggatctccaa ttggtgacgg tccagtcatc aatcggagtg aggattcagc
     4561 tgcttctcga ggccgcacat caatcggagt gaggatgtac agggtgggct gttccaccat
     4621 caatcggagt gaggatttgc gtcaatgggg cggagttcat caatcggagt gaggatatcg
     4681 aagtcatcga gagcactcat caatcggagt gaggatatac tccacccatt gacgtcacat
     4741 caatcggagt gaggattgga accagaaggg tctcttcatc aatcggagtg aggataccct
     4801 atgggcgcgc cgtcgctggc aataatagcg ggcggacgca tgtcatgaga ttattggaaa
     4861 ccaccagaat cgaatataaa aggcgaacac ctttcccaat tttggtttcc cctgacccaa
     4921 agactttaaa tttaatttat ttgtccctat ttcaatcaat tgaacaacta tcaaaacaca
     4981 gaattcatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
     5041 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
     5101 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
     5161 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
     5221 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
     5281 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
     5341 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
     5401 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
     5461 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
     5521 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
     5581 aaccactacc tgagcaccca gagcgccctg agcaaagacc ccaacgagaa gcgcgatcac
     5641 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     5701 aaggccgcgg cggccgccag ctttctagaa caaaaactca tctcagaaga ggatctgaat
     5761 agcgccgtcg accatcatca tcatcatcat tgaccggtct tgctagattc taatcaagag
     5821 gatgtcagaa tgccatttgc ctgagagatg caggcttcat ttttgatact tttttatttg
     5881 taacctatat agtataggat tttttttgtc attttgtt
//
