LOCUS       sequence-147729-        7903 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  255B. Promoter: scTEF1, minimal Promoter: GAP. Figure 5. Expresses 
            rHGH and IFNa2b..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7903)
  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 7903)
  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: PP363
FEATURES             Location/Qualifiers
     source          1..7903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        15..426
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        434..481
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             500..874
                     /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      940..1187
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(956..974)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        1202..2140
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             join(2141..2197,2198..2395)
                     /codon_start=1
                     /gene="MF-alpha-1"
                     /product="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
                     /label=alpha-factor secretion signal
                     /note="Cleavage by the Kex2 protease occurs after the 
                     dibasic KR sequence."
                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR"
     terminator      2915..3161
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     rep_origin      complement(3222..3810)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3302..3321)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    3930..3979
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     promoter        4100..4506
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     CDS             4530..4550
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5769..5918
                     /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             5925..5951
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      5993..6239
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(6805..6861,6862..7059)
                     /codon_start=1
                     /gene="MF-alpha-1"
                     /product="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
                     /label=alpha-factor secretion signal
                     /note="Cleavage by the Kex2 protease occurs after the 
                     dibasic KR sequence."
                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR"
     terminator      7657..7903
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
ORIGIN
        1 ccttcgtttg tgcggatccc ccacacacca tagcttcaaa atgtttctac tcctttttta
       61 ctcttccaga ttttctcgga ctccgcgcat cgccgtacca cttcaaaaca cccaagcaca
      121 gcatactaaa ttttccctct ttcttcctct agggtgtcgt taattacccg tactaaaggt
      181 ttggaaaaga aaaaagagac cgcctcgttt ctttttcttc gtcgaaaaag gcaataaaaa
      241 tttttatcac gtttcttttt cttgaaattt ttttttttag tttttttctc tttcagtgac
      301 ctccattgat atttaagtta ataaacggtc ttcaatttct caagtttcag tttcattttt
      361 cttgttctat tacaactttt tttacttctt gttcattaga aagaaagcat agcaatctaa
      421 tctaaggggc ggtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga
      481 caaggtgagg aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
      541 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
      601 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
      661 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
      721 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
      781 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
      841 ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtc cgacggcggc ccacgggtcc
      901 caggcctcgg agatccgtcc cccttttcct ttgtcgatat catgtaatta gttatgtcac
      961 gcttacattc acgccctccc cccacatccg ctctaaccga aaaggaagga gttagacaac
     1021 ctgaagtcta ggtccctatt tattttttta tagttatgtt agtattaaga acgttattta
     1081 tatttcaaat ttttcttttt tttctgtaca gacgcgtgta cgcatgtaac attatactga
     1141 aaaccttgct tgagaaggtt ttgggacgct cgaaggcttt aatttgcaag ctggagacca
     1201 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag
     1261 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt
     1321 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc
     1381 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta
     1441 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta
     1501 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg
     1561 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct
     1621 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg
     1681 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcggca taccgtttgt
     1741 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct
     1801 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct
     1861 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact
     1921 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat
     1981 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt
     2041 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga
     2101 caacttgaga agatcaaaaa acaactaatt attcgaaacg atgagatttc cttcaatttt
     2161 tactgctgtt ttattcgcag catcctccgc attagctgct ccagtcaaca ctacaacaga
     2221 agatgaaacg gcacaaattc cggctgaagc tgtcatcggt tactcagatt tagaagggga
     2281 tttcgatgtt gctgttttgc cattttccaa cagcacaaat aacgggttat tgtttataaa
     2341 tactactatt gccagcattg ctgctaaaga agaaggggta tctctcgaga agagatgcga
     2401 cttgcctcaa actcattcac tgggttctcg taggacattg atgcttcttg cacagatgag
     2461 aaggatctca ttattttctt gtctaaaaga caggcacgat ttcggattcc cacaagagga
     2521 atttggaaat caatttcaaa aagctgaaac aattcctgtt ttgcatgaga tgatacagca
     2581 aatatttaac ttgttttcaa ccaaagattc ctcagcagcc tgggacgaaa cccttttgga
     2641 caaattttac actgaactat atcaacagct taacgatttg gaggcttgcg taattcaagg
     2701 agtgggtgtt actgaaaccc ccctaatgaa tgaagactct atactggccg tcagaaaata
     2761 ctttcaaaga attactttgt acttgaaaga aaaaaagtat tcaccttgcg cctgggaggt
     2821 cgttagagcc gaaattatga gaagtttttc tttgagtaca aacctgcagg agtcattgcg
     2881 atcaaaagaa tgaaccggtc ttgctagatt ctaatcaaga ggatgtcaga atgccatttg
     2941 cctgagagat gcaggcttca tttttgatac ttttttattt gtaacctata tagtatagga
     3001 ttttttttgt cattttgttt cttctcgtac gagcttgctc ctgatcagcc tatctcgcag
     3061 ctgatgaata tcttgtggta ggggtttggg aaaatcattc gagtttgatg tttttcttgg
     3121 tatttcccac tcctcttcag agtacagaag attaagtgag acatgtgagc aaaaggccag
     3181 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     3241 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     3301 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     3361 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc
     3421 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     3481 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     3541 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     3601 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     3661 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     3721 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     3781 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     3841 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatc agatctgagc
     3901 tcgtttggcc gtggccgtgc tcgtcctcgt cggccggctt gtcgacgacg gcggtctccg
     3961 tcgtcaggat catccgggcc acaagcttgc tgacagaagc ctcaagaaaa aaaaaattct
     4021 tcttcgacta tgctggaggc agagatgatc gagccggtag ttaactatat atagctaaat
     4081 tggttccatc acctcgagtc cacacaccat agcttcaaaa tgtttctact ccttttttac
     4141 tcttccagat tttctcggac tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag
     4201 catactaaat ttcccctctt tcttcctcta gggtgtcgtt aattacccgt actaaaggtt
     4261 tggaaaagaa aaaagagacc gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat
     4321 ttttatcacg tttctttttc ttgaaaattt tttttttgat ttttttctct ttcgatgacc
     4381 tcccattgat atttaagtta ataaacggtc ttcaatttct caagtttcag tttcattttt
     4441 cttgttctat tacaactttt tttacttctt gctcattaga aagaaagcat agcaatctaa
     4501 tctaaggact agtaaaaatg cgcggagctc ctaagaaaaa gcgcaaagtc cggccggcat
     4561 ctagacccgg ggagcgcccc ttccagtgtc gcatttgcat gcggaacttt tcgcgccagg
     4621 acaggcttga caggcatacc cgtactcata ccggtgaaaa accgtttcag tgtcggatct
     4681 gtatgcgaaa tttctcccag aaggagcact tggcggggca tctacgtacg cacaccggcg
     4741 agaagccatt ccaatgccga atatgcatgc gcaacttcag tcgccgcgac aacctgaacc
     4801 ggcacctaaa aacccacctg aggaacatat gcggcggagg cacacctgca gctgcgtcga
     4861 ctctagagga tccatctgct ggagacatga gagctgccaa cctttggcca agcccgctca
     4921 tgatcaaacg ctctaagaag aacagcctgg ccttgtccct gacggccgac cagatggtca
     4981 gtgccttgtt ggatgctgag ccccccatac tctattccga gtatgatcct accagaccct
     5041 tcagtgaagc ttcgatgatg ggcttactga ccaacctggc agacagggag ctggttcaca
     5101 tgatcaactg ggcgaagagg gtgccaggct ttgtggattt gaccctccat gatcaggtcc
     5161 accttctaga atgtgcctgg ctagagatcc tgatgattgg tctcgtctgg cgctccatgg
     5221 agcacccagt gaagctactg tttgctccta acttgctctt ggacaggaac cagggaaaat
     5281 gtgtagaggg catggtggag atcttcgaca tgctgctggc tacatcatct cggttccgca
     5341 tgatgaatct gcagggagag gagtttgtgt gcctcaaatc tattattttg cttaattctg
     5401 gagtgtacac atttctgtcc agcaccctga agtctctgga agagaaggac catatccacc
     5461 gagtcctgga caagatcaca gacactttga tccacctgat ggccaaggca ggcctgaccc
     5521 tgcagcagca gcaccagcgg ctggcccagc tcctcctcat cctctcccac atcaggcaca
     5581 tgagtaacaa aggcatggag catctgtaca gcatgaagtg caagaacgtg gtgcccctct
     5641 atgacctgct gctggagatg ctggacgccc accgcctaca tgcgcccact agccgtggag
     5701 gggcatccgt ggaggagacg gaccaaagcc acttggccac tgcgggctct acttcatcgc
     5761 ctagggccga cgcgctggac gatttcgatc tcgacatgct gggttctgat gccctcgatg
     5821 actttgacct ggatatgttg ggaagcgacg cattggatga ctttgatctg gacatgctcg
     5881 gctccgatgc tctggacgat ttcgatctcg atatgttaat taactacccg tacgacgttc
     5941 cggactacgc ttcttgaggt accatcggta gaccggtctt gctagattct aatcaagagg
     6001 atgtcagaat gccatttgcc tgagagatgc aggcttcatt tttgatactt ttttatttgt
     6061 aacctatata gtataggatt ttttttgtca ttttgtttct tctcgtacga gcttgctcct
     6121 gatcagccta tctcgcagct gatgaatatc ttgtggtagg ggtttgggaa aatcattcga
     6181 gtttgatgtt tttcttggta tttcccactc ctcttcagag tacagaagat taagtgagag
     6241 ctagcatatc atatagaagt catcgcggca gatcaattca tcaatcggag tgaggatatt
     6301 gcgagtttac caccatcaat cggagtgagg atctccaatt ggtgacggtc cagtcatcaa
     6361 tcggagtgag gattcagctg cttctcgagg ccgcacatca atcggagtga ggatgtacag
     6421 ggtgggctgt tccaccatca atcggagtga ggatttgcgt caatggggcg gagttcatca
     6481 atcggagtga ggatatcgaa gtcatcgaga gcactcatca atcggagtga ggatatactc
     6541 cacccattga cgtcacatca atcggagtga ggattggaac cagaagggtc tcttcatcaa
     6601 tcggagtgag gataccctat gggcgcgccg tcgctggcaa taatagcggg cggacgcatg
     6661 tcatgagatt attggaaacc accagaatcg aatataaaag gcgaacacct ttcccaattt
     6721 tggtttcccc tgacccaaag actttaaatt taatttattt gtccctattt caatcaattg
     6781 aacaactatc aaaacacaga attcatgaga tttccttcaa tttttactgc tgttttattc
     6841 gcagcatcct ccgcattagc tgctccagtc aacactacaa cagaagatga aacggcacaa
     6901 attccggctg aagctgtcat cggttactca gatttagaag gggatttcga tgttgctgtt
     6961 ttgccatttt ccaacagcac aaataacggg ttattgttta taaatactac tattgccagc
     7021 attgctgcta aagaagaagg ggtatctctc gagaagagat tcccaaccat tcccttatct
     7081 agactttttg acaacgctat gctccgcgcc catcgtctgc accagctggc ctttgacacc
     7141 taccaggagt ttgaagaagc ctatatccca aaggaacaga agtattcatt cctgcagaac
     7201 ccccagacct ccctctgttt ctcagagtct attccgacac cctccaacag ggaggaaaca
     7261 caacagaaat ccaacctaga gctgctccgc atctccctgc tgctcatcca gtcgtggctg
     7321 gagcccgtgc agttcctcag gagtgtcttc gccaacagcc tggtgtacgg cgcctctgac
     7381 agcaacgtct atgacctcct aaaggaccta gaggaaggca tccaaacgct gatggggagg
     7441 ctggaagatg gcagcccccg gactgggcag atcttcaagc agacctacag caagttcgac
     7501 acaaactcac acaacgatga cgcactactc aagaactacg ggctgctcta ctgcttcagg
     7561 aaggacatgg acaaggtcga gacattcctg cgcatcgtgc agtgccgctc tgtggagggc
     7621 agctgtggct tctagaccgg tcttgctaga ttctaatcaa gaggatgtca gaatgccatt
     7681 tgcctgagag atgcaggctt catttttgat acttttttat ttgtaaccta tatagtatag
     7741 gatttttttt gtcattttgt ttcttctcgt acgagcttgc tcctgatcag cctatctcgc
     7801 agctgatgaa tatcttgtgg taggggtttg ggaaaatcat tcgagtttga tgtttttctt
     7861 ggtatttccc actcctcttc agagtacaga agattaagtg aga
//
