LOCUS       sequence-147728-        8595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  245B. Promoter: GAP, 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 8595)
  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 8595)
  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: PP326
FEATURES             Location/Qualifiers
     source          1..8595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(19..607)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(99..118)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    727..776
                     /label=Bxb1 attB
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="attB site for the Mycopbacteriophage Bxb1 integrase 
                     (Xu et al., 2013)"
     promoter        1501..1977
                     /label=GAP promoter
                     /note="promoter for Pichia pastoris 
                     glyceraldehyde-3-phosphate dehydrogenase"
     CDS             2008..2028
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3247..3396
                     /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             3403..3429
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     terminator      3471..3717
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             join(4296..4352,4353..4550)
                     /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      5146..5392
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     promoter        5407..5818
                     /gene="S. cerevisiae TEF1"
                     /label=TEF1 promoter
                     /note="promoter for EF-1-alpha"
     promoter        5826..5873
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             5892..6266
                     /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      6332..6579
                     /gene="S. cerevisiae CYC1"
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     primer_bind     complement(6348..6366)
                     /label=CYC1
                     /note="CYC1 transcription termination signal, reverse 
                     primer"
     promoter        6594..7532
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             join(7533..7589,7590..7787)
                     /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      8307..8553
                     /gene="Pichia pastoris AOX1"
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
ORIGIN
        1 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
       61 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
      121 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
      181 ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc
      241 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
      301 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
      361 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
      421 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
      481 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
      541 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
      601 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
      661 acgttaaggg attttggtca tgagatcaga tctgagctcg tttggccgtg gccgtgctcg
      721 tcctcgtcgg ccggcttgtc gacgacggcg gtctccgtcg tcaggatcat ccgggccaca
      781 agcttgctga cagaagcctc aagaaaaaaa aaattcttct tcgactatgc tggaggcaga
      841 gatgatcgag ccggtagtta actatatata gctaaattgg ttccatcacc tcgagtcaat
      901 tcttgattta gtatacacat aaccaaattt ggatcaagtt tgaagtaaaa ctttaacttc
      961 agctccttac atttgcacta agatctctgc tactctggtc ccaagtgaac caccttttgg
     1021 accctattga ccggacctta acttgccaaa cctaaacgct taatgcctca gacgttttaa
     1081 tgcctctcaa cacctccaag gttgctttct tgagcatgcc tactagggac tttaacgaac
     1141 tgtggggttg cagacagttt caggcgtgtc ccgaccaata tggcctacta gactctctga
     1201 aaaatcacag ttttccagta gttccgatca aattaccatc gaaatggtcc cataaacgga
     1261 catttgacat ccgttcctga attatagtct tccaccgtgg atcatggtgt tccttttttt
     1321 cccaaagaat atcagcatcc cttaactacg ttaggtcagt gatgacaatg gaccaaattg
     1381 ttgcaaggtt tttctttttc tttcatcggc acatttcagc ctcacatgcg actattatcg
     1441 atcaatgaaa tccatcaaga ttgaaatctt aaaattgccc ctttcacttg acaggatcct
     1501 tttttgtaga aatgtcttgg tgtcctcgtc caatcaggta gccatctctg aaatatctgg
     1561 ctccgttgca actccgaacg acctgctggc aacgtaaaat tctccggggt aaaactttaa
     1621 tgtggagtaa tggaaccaga aacgtctctt cccttctctc tccttccacc gcccgttacc
     1681 gtccctagga aattttactc tgctggagag cttcttctac ggcccccttg cagcaatgct
     1741 cttcccagca ttacgttgcg ggtaaaacgg aggtcgtgta cccgacctag cagcccaggg
     1801 atggaaaagt cccggccgtc gctggcaata atagcgggcg gacgcatgtc atgagattat
     1861 tggaaaccac cagaatcgaa tataaaaggc gaacaccttt cccaattttg gtttcccctg
     1921 acccaaagac tttaaattta atttatttgt ccctatttca atcaattgaa caactatcaa
     1981 aacacactag taaaaatgcg cggagctcct aagaaaaagc gcaaagtccg gccggcatct
     2041 agacccgggg agcgcccctt ccagtgtcgc atttgcatgc ggaacttttc gcgccaggac
     2101 aggcttgaca ggcatacccg tactcatacc ggtgaaaaac cgtttcagtg tcggatctgt
     2161 atgcgaaatt tctcccagaa ggagcacttg gcggggcatc tacgtacgca caccggcgag
     2221 aagccattcc aatgccgaat atgcatgcgc aacttcagtc gccgcgacaa cctgaaccgg
     2281 cacctaaaaa cccacctgag gaacatatgc ggcggaggca cacctgcagc tgcgtcgact
     2341 ctagaggatc catctgctgg agacatgaga gctgccaacc tttggccaag cccgctcatg
     2401 atcaaacgct ctaagaagaa cagcctggcc ttgtccctga cggccgacca gatggtcagt
     2461 gccttgttgg atgctgagcc ccccatactc tattccgagt atgatcctac cagacccttc
     2521 agtgaagctt cgatgatggg cttactgacc aacctggcag acagggagct ggttcacatg
     2581 atcaactggg cgaagagggt gccaggcttt gtggatttga ccctccatga tcaggtccac
     2641 cttctagaat gtgcctggct agagatcctg atgattggtc tcgtctggcg ctccatggag
     2701 cacccagtga agctactgtt tgctcctaac ttgctcttgg acaggaacca gggaaaatgt
     2761 gtagagggca tggtggagat cttcgacatg ctgctggcta catcatctcg gttccgcatg
     2821 atgaatctgc agggagagga gtttgtgtgc ctcaaatcta ttattttgct taattctgga
     2881 gtgtacacat ttctgtccag caccctgaag tctctggaag agaaggacca tatccaccga
     2941 gtcctggaca agatcacaga cactttgatc cacctgatgg ccaaggcagg cctgaccctg
     3001 cagcagcagc accagcggct ggcccagctc ctcctcatcc tctcccacat caggcacatg
     3061 agtaacaaag gcatggagca tctgtacagc atgaagtgca agaacgtggt gcccctctat
     3121 gacctgctgc tggagatgct ggacgcccac cgcctacatg cgcccactag ccgtggaggg
     3181 gcatccgtgg aggagacgga ccaaagccac ttggccactg cgggctctac ttcatcgcct
     3241 agggccgacg cgctggacga tttcgatctc gacatgctgg gttctgatgc cctcgatgac
     3301 tttgacctgg atatgttggg aagcgacgca ttggatgact ttgatctgga catgctcggc
     3361 tccgatgctc tggacgattt cgatctcgat atgttaatta actacccgta cgacgttccg
     3421 gactacgctt cttgaggtac catcggtaga ccggtcttgc tagattctaa tcaagaggat
     3481 gtcagaatgc catttgcctg agagatgcag gcttcatttt tgatactttt ttatttgtaa
     3541 cctatatagt ataggatttt ttttgtcatt ttgtttcttc tcgtacgagc ttgctcctga
     3601 tcagcctatc tcgcagctga tgaatatctt gtggtagggg tttgggaaaa tcattcgagt
     3661 ttgatgtttt tcttggtatt tcccactcct cttcagagta cagaagatta agtgagagct
     3721 agcatatcat atagaagtca tcgcggcaga tcaattcatc aatcggagtg aggatattgc
     3781 gagtttacca ccatcaatcg gagtgaggat ctccaattgg tgacggtcca gtcatcaatc
     3841 ggagtgagga ttcagctgct tctcgaggcc gcacatcaat cggagtgagg atgtacaggg
     3901 tgggctgttc caccatcaat cggagtgagg atttgcgtca atggggcgga gttcatcaat
     3961 cggagtgagg atatcgaagt catcgagagc actcatcaat cggagtgagg atatactcca
     4021 cccattgacg tcacatcaat cggagtgagg attggaacca gaagggtctc ttcatcaatc
     4081 ggagtgagga taccctatgg gcgcgcctaa cccctacttg acagcaatat ataaacagaa
     4141 ggaagctgcc ctgtcttaaa cctttttttt tatcatcatt attagcttac tttcataatt
     4201 gcgactggtt ccaattgaca agcttttgat tttaacgact tttaacgaca acttgagaag
     4261 atcaaaaaac aactaattat tcgaaacgcg aattcatgag atttccttca atttttactg
     4321 ctgttttatt cgcagcatcc tccgcattag ctgctccagt caacactaca acagaagatg
     4381 aaacggcaca aattccggct gaagctgtca tcggttactc agatttagaa ggggatttcg
     4441 atgttgctgt tttgccattt tccaacagca caaataacgg gttattgttt ataaatacta
     4501 ctattgccag cattgctgct aaagaagaag gggtatctct cgagaagaga ttcccaacca
     4561 ttcccttatc tagacttttt gacaacgcta tgctccgcgc ccatcgtctg caccagctgg
     4621 cctttgacac ctaccaggag tttgaagaag cctatatccc aaaggaacag aagtattcat
     4681 tcctgcagaa cccccagacc tccctctgtt tctcagagtc tattccgaca ccctccaaca
     4741 gggaggaaac acaacagaaa tccaacctag agctgctccg catctccctg ctgctcatcc
     4801 agtcgtggct ggagcccgtg cagttcctca ggagtgtctt cgccaacagc ctggtgtacg
     4861 gcgcctctga cagcaacgtc tatgacctcc taaaggacct agaggaaggc atccaaacgc
     4921 tgatggggag gctggaagat ggcagccccc ggactgggca gatcttcaag cagacctaca
     4981 gcaagttcga cacaaactca cacaacgatg acgcactact caagaactac gggctgctct
     5041 actgcttcag gaaggacatg gacaaggtcg agacattcct gcgcatcgtg cagtgccgct
     5101 ctgtggaggg cagctgtggc ttctagcggt cttgctagat tctaatcaag aggatgtcag
     5161 aatgccattt gcctgagaga tgcaggcttc atttttgata cttttttatt tgtaacctat
     5221 atagtatagg attttttttg tcattttgtt tcttctcgta cgagcttgct cctgatcagc
     5281 ctatctcgca gctgatgaat atcttgtggt aggggtttgg gaaaatcatt cgagtttgat
     5341 gtttttcttg gtatttccca ctcctcttca gagtacagaa gattaagtga gaccttcgtt
     5401 tgtgcggatc ccccacacac catagcttca aaatgtttct actccttttt tactcttcca
     5461 gattttctcg gactccgcgc atcgccgtac cacttcaaaa cacccaagca cagcatacta
     5521 aattttccct ctttcttcct ctagggtgtc gttaattacc cgtactaaag gtttggaaaa
     5581 gaaaaaagag accgcctcgt ttctttttct tcgtcgaaaa aggcaataaa aatttttatc
     5641 acgtttcttt ttcttgaaat tttttttttt agtttttttc tctttcagtg acctccattg
     5701 atatttaagt taataaacgg tcttcaattt ctcaagtttc agtttcattt ttcttgttct
     5761 attacaactt tttttacttc ttgttcatta gaaagaaagc atagcaatct aatctaaggg
     5821 gcggtgttga caattaatca tcggcatagt atatcggcat agtataatac gacaaggtga
     5881 ggaactaaac catggccaag ttgaccagtg ccgttccggt gctcaccgcg cgcgacgtcg
     5941 ccggagcggt cgagttctgg accgaccggc tcgggttctc ccgggacttc gtggaggacg
     6001 acttcgccgg tgtggtccgg gacgacgtga ccctgttcat cagcgcggtc caggaccagg
     6061 tggtgccgga caacaccctg gcctgggtgt gggtgcgcgg cctggacgag ctgtacgccg
     6121 agtggtcgga ggtcgtgtcc acgaacttcc gggacgcctc cgggccggcc atgaccgaga
     6181 tcggcgagca gccgtggggg cgggagttcg ccctgcgcga cccggccggc aactgcgtgc
     6241 acttcgtggc cgaggagcag gactgacacg tccgacggcg gcccacgggt cccaggcctc
     6301 ggagatccgt cccccttttc ctttgtcgat atcatgtaat tagttatgtc acgcttacat
     6361 tcacgccctc cccccacatc cgctctaacc gaaaaggaag gagttagaca acctgaagtc
     6421 taggtcccta tttatttttt tatagttatg ttagtattaa gaacgttatt tatatttcaa
     6481 atttttcttt tttttctgta cagacgcgtg tacgcatgta acattatact gaaaaccttg
     6541 cttgagaagg ttttgggacg ctcgaaggct ttaatttgca agctggagac caagatctaa
     6601 catccaaaga cgaaaggttg aatgaaacct ttttgccatc cgacatccac aggtccattc
     6661 tcacacataa gtgccaaacg caacaggagg ggatacacta gcagcagacc gttgcaaacg
     6721 caggacctcc actcctcttc tcctcaacac ccacttttgc catcgaaaaa ccagcccagt
     6781 tattgggctt gattggagct cgctcattcc aattccttct attaggctac taacaccatg
     6841 actttattag cctgtctatc ctggcccccc tggcgaggtt catgtttgtt tatttccgaa
     6901 tgcaacaagc tccgcattac acccgaacat cactccagat gagggctttc tgagtgtggg
     6961 gtcaaatagt ttcatgttcc ccaaatggcc caaaactgac agtttaaacg ctgtcttgga
     7021 acctaatatg acaaaagcgt gatctcatcc aagatgaact aagtttggtt cgttgaaatg
     7081 ctaacggcca gttggtcaaa aagaaacttc caaaagtcgg cataccgttt gtcttgtttg
     7141 gtattgattg acgaatgctc aaaaataatc tcattaatgc ttagcgcagt ctctctatcg
     7201 cttctgaacc ccggtgcacc tgtgccgaaa cgcaaatggg gaaacacccg ctttttggat
     7261 gattatgcat tgtctccaca ttgtatgctt ccaagattct ggtgggaata ctgctgatag
     7321 cctaacgttc atgatcaaaa tttaactgtt ctaaccccta cttgacagca atatataaac
     7381 agaaggaagc tgccctgtct taaacctttt tttttatcat cattattagc ttactttcat
     7441 aattgcgact ggttccaatt gacaagcttt tgattttaac gacttttaac gacaacttga
     7501 gaagatcaaa aaacaactaa ttattcgaaa cgatgagatt tccttcaatt tttactgctg
     7561 ttttattcgc agcatcctcc gcattagctg ctccagtcaa cactacaaca gaagatgaaa
     7621 cggcacaaat tccggctgaa gctgtcatcg gttactcaga tttagaaggg gatttcgatg
     7681 ttgctgtttt gccattttcc aacagcacaa ataacgggtt attgtttata aatactacta
     7741 ttgccagcat tgctgctaaa gaagaagggg tatctctcga gaagagatgc gacttgcctc
     7801 aaactcattc actgggttct cgtaggacat tgatgcttct tgcacagatg agaaggatct
     7861 cattattttc ttgtctaaaa gacaggcacg atttcggatt cccacaagag gaatttggaa
     7921 atcaatttca aaaagctgaa acaattcctg ttttgcatga gatgatacag caaatattta
     7981 acttgttttc aaccaaagat tcctcagcag cctgggacga aacccttttg gacaaatttt
     8041 acactgaact atatcaacag cttaacgatt tggaggcttg cgtaattcaa ggagtgggtg
     8101 ttactgaaac ccccctaatg aatgaagact ctatactggc cgtcagaaaa tactttcaaa
     8161 gaattacttt gtacttgaaa gaaaaaaagt attcaccttg cgcctgggag gtcgttagag
     8221 ccgaaattat gagaagtttt tctttgagta caaacctgca ggagtcattg cgatcaaaag
     8281 aatgaaccgg tcttgctaga ttctaatcaa gaggatgtca gaatgccatt tgcctgagag
     8341 atgcaggctt catttttgat acttttttat ttgtaaccta tatagtatag gatttttttt
     8401 gtcattttgt ttcttctcgt acgagcttgc tcctgatcag cctatctcgc agctgatgaa
     8461 tatcttgtgg taggggtttg ggaaaatcat tcgagtttga tgtttttctt ggtatttccc
     8521 actcctcttc agagtacaga agattaagtg agacatgtga gcaaaaggcc agcaaaaggc
     8581 caggaaccgt aaaaa
//
