LOCUS       sequence-147727-        8719 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  246B. Promoter: GAP6, minimal Promoter: AOX1. Figure 5. Expresses 
            rHGH and IFNa2b..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8719)
  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 8719)
  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: PP324
FEATURES             Location/Qualifiers
     source          1..8719
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        223..1161
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             join(1162..1218,1219..1416)
                     /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      1936..2182
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     rep_origin      complement(2243..2831)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2323..2342)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    2951..3000
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     protein_bind    4163..4181
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    4184..4202
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     CDS             4272..4292
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5511..5660
                     /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             5667..5693
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      5735..5981
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(6560..6616,6617..6814)
                     /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"
     CDS             7408..7437
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             7453..7470
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      7494..7740
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        7755..8166
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        8174..8221
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             8240..8614
                     /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      join(8680..8719,1..208)
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(8696..8714)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
ORIGIN
        1 ccccacatcc gctctaaccg aaaaggaagg agttagacaa cctgaagtct aggtccctat
       61 ttattttttt atagttatgt tagtattaag aacgttattt atatttcaaa tttttctttt
      121 ttttctgtac agacgcgtgt acgcatgtaa cattatactg aaaaccttgc ttgagaaggt
      181 tttgggacgc tcgaaggctt taatttgcaa gctggagacc aagatctaac atccaaagac
      241 gaaaggttga atgaaacctt tttgccatcc gacatccaca ggtccattct cacacataag
      301 tgccaaacgc aacaggaggg gatacactag cagcagaccg ttgcaaacgc aggacctcca
      361 ctcctcttct cctcaacacc cacttttgcc atcgaaaaac cagcccagtt attgggcttg
      421 attggagctc gctcattcca attccttcta ttaggctact aacaccatga ctttattagc
      481 ctgtctatcc tggcccccct ggcgaggttc atgtttgttt atttccgaat gcaacaagct
      541 ccgcattaca cccgaacatc actccagatg agggctttct gagtgtgggg tcaaatagtt
      601 tcatgttccc caaatggccc aaaactgaca gtttaaacgc tgtcttggaa cctaatatga
      661 caaaagcgtg atctcatcca agatgaacta agtttggttc gttgaaatgc taacggccag
      721 ttggtcaaaa agaaacttcc aaaagtcggc ataccgtttg tcttgtttgg tattgattga
      781 cgaatgctca aaaataatct cattaatgct tagcgcagtc tctctatcgc ttctgaaccc
      841 cggtgcacct gtgccgaaac gcaaatgggg aaacacccgc tttttggatg attatgcatt
      901 gtctccacat tgtatgcttc caagattctg gtgggaatac tgctgatagc ctaacgttca
      961 tgatcaaaat ttaactgttc taacccctac ttgacagcaa tatataaaca gaaggaagct
     1021 gccctgtctt aaaccttttt ttttatcatc attattagct tactttcata attgcgactg
     1081 gttccaattg acaagctttt gattttaacg acttttaacg acaacttgag aagatcaaaa
     1141 aacaactaat tattcgaaac gatgagattt ccttcaattt ttactgctgt tttattcgca
     1201 gcatcctccg cattagctgc tccagtcaac actacaacag aagatgaaac ggcacaaatt
     1261 ccggctgaag ctgtcatcgg ttactcagat ttagaagggg atttcgatgt tgctgttttg
     1321 ccattttcca acagcacaaa taacgggtta ttgtttataa atactactat tgccagcatt
     1381 gctgctaaag aagaaggggt atctctcgag aagagatgcg acttgcctca aactcattca
     1441 ctgggttctc gtaggacatt gatgcttctt gcacagatga gaaggatctc attattttct
     1501 tgtctaaaag acaggcacga tttcggattc ccacaagagg aatttggaaa tcaatttcaa
     1561 aaagctgaaa caattcctgt tttgcatgag atgatacagc aaatatttaa cttgttttca
     1621 accaaagatt cctcagcagc ctgggacgaa acccttttgg acaaatttta cactgaacta
     1681 tatcaacagc ttaacgattt ggaggcttgc gtaattcaag gagtgggtgt tactgaaacc
     1741 cccctaatga atgaagactc tatactggcc gtcagaaaat actttcaaag aattactttg
     1801 tacttgaaag aaaaaaagta ttcaccttgc gcctgggagg tcgttagagc cgaaattatg
     1861 agaagttttt ctttgagtac aaacctgcag gagtcattgc gatcaaaaga atgaaccggt
     1921 cttgctagat tctaatcaag aggatgtcag aatgccattt gcctgagaga tgcaggcttc
     1981 atttttgata cttttttatt tgtaacctat atagtatagg attttttttg tcattttgtt
     2041 tcttctcgta cgagcttgct cctgatcagc ctatctcgca gctgatgaat atcttgtggt
     2101 aggggtttgg gaaaatcatt cgagtttgat gtttttcttg gtatttccca ctcctcttca
     2161 gagtacagaa gattaagtga gacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     2221 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     2281 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     2341 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     2401 ccgcctttct cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca
     2461 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     2521 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     2581 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     2641 cagagttctt gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct
     2701 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     2761 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     2821 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     2881 actcacgtta agggattttg gtcatgagat cagatctgag ctcgtttggc cgtggccgtg
     2941 ctcgtcctcg tcggccggct tgtcgacgac ggcggtctcc gtcgtcagga tcatccgggc
     3001 cacaagcttg ctgacagaag cctcaagaaa aaaaaaattc ttcttcgact atgctggagg
     3061 cagagatgat cgagccggta gttaactata tatagctaaa ttggttccat cacctcgagt
     3121 ctattcttga tttagtatac acataaccaa atttggatca agtttgaagt aaaactttaa
     3181 cttcagctcc ttacatttgc actaagatct ctgctactct ggtcccaagt gaaccacctt
     3241 ttggacccta ttgaccggac cttaacttgc caaacctaaa cgcttaatgc ctcagacgtt
     3301 ttaatgcctc tcaacacctc caaggttgct ttcttgagca tgcctactag ggactttaac
     3361 gaactgtggg gttgcagaca gtttcaggcg tgtcccgacc aatatggcct actagactct
     3421 ctgaaaaatc acagttttcc agtagttccg atcaaattac catcgaaatg gtcccataaa
     3481 cggacatttg acatccgttc ctgaattata gtcttccacc gtggatcatg gtgttccttt
     3541 ttttcccaaa gaatatcagc atcccttaac tacgttaggt cagtgatgac aatggaccaa
     3601 attgttgcaa ggtttttctt tttctttcat cggcacattt cagcctcaca tgcgactatt
     3661 atcgatcaat gaaatccatc aagattgaaa tcttaaaatt gcccctttca cttgacagga
     3721 tccttttttg tagaaatgtc ttggtgtcct cgtccaatca ggtagccatc tctgaaatat
     3781 ctggctccgt tgcaactccg aacgacctgc tggcaacgta aaattctccg gggtaaaact
     3841 ttaatgtgga gtaatggaac cagaaacgtc tcttcccttc tctctccttc caccgcccgt
     3901 taccgtccct aggaaatttt actctgctgg agagcttctt ctacggcccc cttgcagcaa
     3961 tgctcttccc agcattacgt tgcgggtaaa acggaggtcg tgtacccgac ctagcagccc
     4021 agggatggaa aagtcccggc cgtcgctggc aataatagcg ggcggacgca tgtcatgaga
     4081 ttattggaaa ccaccagaat cgaatataaa aggcgaacac ctttcccaat tttggtttct
     4141 cctgacccaa agactttaaa tttccctatc agtgatagag atctccctat cagtgataga
     4201 gataatttat ttgtccctat ttcaatcaat tgaacaacta tcaaaacaca ctagtaaaaa
     4261 tgcgcggagc tcctaagaaa aagcgcaaag tccggccggc atctagaccc ggggagcgcc
     4321 ccttccagtg tcgcatttgc atgcggaact tttcgcgcca ggacaggctt gacaggcata
     4381 cccgtactca taccggtgaa aaaccgtttc agtgtcggat ctgtatgcga aatttctccc
     4441 agaaggagca cttggcgggg catctacgta cgcacaccgg cgagaagcca ttccaatgcc
     4501 gaatatgcat gcgcaacttc agtcgccgcg acaacctgaa ccggcaccta aaaacccacc
     4561 tgaggaacat atgcggcgga ggcacacctg cagctgcgtc gactctagag gatccatctg
     4621 ctggagacat gagagctgcc aacctttggc caagcccgct catgatcaaa cgctctaaga
     4681 agaacagcct ggccttgtcc ctgacggccg accagatggt cagtgccttg ttggatgctg
     4741 agccccccat actctattcc gagtatgatc ctaccagacc cttcagtgaa gcttcgatga
     4801 tgggcttact gaccaacctg gcagacaggg agctggttca catgatcaac tgggcgaaga
     4861 gggtgccagg ctttgtggat ttgaccctcc atgatcaggt ccaccttcta gaatgtgcct
     4921 ggctagagat cctgatgatt ggtctcgtct ggcgctccat ggagcaccca gtgaagctac
     4981 tgtttgctcc taacttgctc ttggacagga accagggaaa atgtgtagag ggcatggtgg
     5041 agatcttcga catgctgctg gctacatcat ctcggttccg catgatgaat ctgcagggag
     5101 aggagtttgt gtgcctcaaa tctattattt tgcttaattc tggagtgtac acatttctgt
     5161 ccagcaccct gaagtctctg gaagagaagg accatatcca ccgagtcctg gacaagatca
     5221 cagacacttt gatccacctg atggccaagg caggcctgac cctgcagcag cagcaccagc
     5281 ggctggccca gctcctcctc atcctctccc acatcaggca catgagtaac aaaggcatgg
     5341 agcatctgta cagcatgaag tgcaagaacg tggtgcccct ctatgacctg ctgctggaga
     5401 tgctggacgc ccaccgccta catgcgccca ctagccgtgg aggggcatcc gtggaggaga
     5461 cggaccaaag ccacttggcc actgcgggct ctacttcatc gcctagggcc gacgcgctgg
     5521 acgatttcga tctcgacatg ctgggttctg atgccctcga tgactttgac ctggatatgt
     5581 tgggaagcga cgcattggat gactttgatc tggacatgct cggctccgat gctctggacg
     5641 atttcgatct cgatatgtta attaactacc cgtacgacgt tccggactac gcttcttgag
     5701 gtaccatcgg tagaccggtc ttgctagatt ctaatcaaga ggatgtcaga atgccatttg
     5761 cctgagagat gcaggcttca tttttgatac ttttttattt gtaacctata tagtatagga
     5821 ttttttttgt cattttgttt cttctcgtac gagcttgctc ctgatcagcc tatctcgcag
     5881 ctgatgaata tcttgtggta ggggtttggg aaaatcattc gagtttgatg tttttcttgg
     5941 tatttcccac tcctcttcag agtacagaag attaagtgag agctagcata tcatatagaa
     6001 gtcatcgcgg cagatcaatt catcaatcgg agtgaggata ttgcgagttt accaccatca
     6061 atcggagtga ggatctccaa ttggtgacgg tccagtcatc aatcggagtg aggattcagc
     6121 tgcttctcga ggccgcacat caatcggagt gaggatgtac agggtgggct gttccaccat
     6181 caatcggagt gaggatttgc gtcaatgggg cggagttcat caatcggagt gaggatatcg
     6241 aagtcatcga gagcactcat caatcggagt gaggatatac tccacccatt gacgtcacat
     6301 caatcggagt gaggattgga accagaaggg tctcttcatc aatcggagtg aggataccct
     6361 atgggcgcgc ctaaccccta cttgacagca atatataaac agaaggaagc tgccctgtct
     6421 taaacctttt tttttatcat cattattagc ttactttcat aattgcgact ggttccaatt
     6481 gacaagcttt tgattttaac gacttttaac gacaacttga gaagatcaaa aaacaactaa
     6541 ttattcgaaa cgcgaattca tgagatttcc ttcaattttt actgctgttt tattcgcagc
     6601 atcctccgca ttagctgctc cagtcaacac tacaacagaa gatgaaacgg cacaaattcc
     6661 ggctgaagct gtcatcggtt actcagattt agaaggggat ttcgatgttg ctgttttgcc
     6721 attttccaac agcacaaata acgggttatt gtttataaat actactattg ccagcattgc
     6781 tgctaaagaa gaaggggtat ctctcgagaa gagattccca accattccct tatctagact
     6841 ttttgacaac gctatgctcc gcgcccatcg tctgcaccag ctggcctttg acacctacca
     6901 ggagtttgaa gaagcctata tcccaaagga acagaagtat tcattcctgc agaaccccca
     6961 gacctccctc tgtttctcag agtctattcc gacaccctcc aacagggagg aaacacaaca
     7021 gaaatccaac ctagagctgc tccgcatctc cctgctgctc atccagtcgt ggctggagcc
     7081 cgtgcagttc ctcaggagtg tcttcgccaa cagcctggtg tacggcgcct ctgacagcaa
     7141 cgtctatgac ctcctaaagg acctagagga aggcatccaa acgctgatgg ggaggctgga
     7201 agatggcagc ccccggactg ggcagatctt caagcagacc tacagcaagt tcgacacaaa
     7261 ctcacacaac gatgacgcac tactcaagaa ctacgggctg ctctactgct tcaggaagga
     7321 catggacaag gtcgagacat tcctgcgcat cgtgcagtgc cgctctgtgg agggcagctg
     7381 tggcttctag cggccgccag ctttctagaa caaaaactca tctcagaaga ggatctgaat
     7441 agcgccgtcg accatcatca tcatcatcat tgaccggtct tgctagattc taatcaagag
     7501 gatgtcagaa tgccatttgc ctgagagatg caggcttcat ttttgatact tttttatttg
     7561 taacctatat agtataggat tttttttgtc attttgtttc ttctcgtacg agcttgctcc
     7621 tgatcagcct atctcgcagc tgatgaatat cttgtggtag gggtttggga aaatcattcg
     7681 agtttgatgt ttttcttggt atttcccact cctcttcaga gtacagaaga ttaagtgaga
     7741 ccttcgtttg tgcggatccc ccacacacca tagcttcaaa atgtttctac tcctttttta
     7801 ctcttccaga ttttctcgga ctccgcgcat cgccgtacca cttcaaaaca cccaagcaca
     7861 gcatactaaa ttttccctct ttcttcctct agggtgtcgt taattacccg tactaaaggt
     7921 ttggaaaaga aaaaagagac cgcctcgttt ctttttcttc gtcgaaaaag gcaataaaaa
     7981 tttttatcac gtttcttttt cttgaaattt ttttttttag tttttttctc tttcagtgac
     8041 ctccattgat atttaagtta ataaacggtc ttcaatttct caagtttcag tttcattttt
     8101 cttgttctat tacaactttt tttacttctt gttcattaga aagaaagcat agcaatctaa
     8161 tctaaggggc ggtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga
     8221 caaggtgagg aactaaacca tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg
     8281 cgacgtcgcc ggagcggtcg agttctggac cgaccggctc gggttctccc gggacttcgt
     8341 ggaggacgac ttcgccggtg tggtccggga cgacgtgacc ctgttcatca gcgcggtcca
     8401 ggaccaggtg gtgccggaca acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct
     8461 gtacgccgag tggtcggagg tcgtgtccac gaacttccgg gacgcctccg ggccggccat
     8521 gaccgagatc ggcgagcagc cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa
     8581 ctgcgtgcac ttcgtggccg aggagcagga ctgacacgtc cgacggcggc ccacgggtcc
     8641 caggcctcgg agatccgtcc cccttttcct ttgtcgatat catgtaatta gttatgtcac
     8701 gcttacattc acgccctcc
//
