LOCUS       sequence-316761-        4686 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  E.coli/ P.pastoris shuttle plasmid for episomal expression in 
            P.pastoris.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4686)
  AUTHORS   Pullmann P, Weissenborn MJ
  TITLE     Improving the Heterologous Production of Fungal Peroxygenases 
            through an Episomal Pichia pastoris Promoter and Signal Peptide 
            Shuffling System.
  JOURNAL   ACS Synth Biol. 2021 Jun 2. doi: 10.1021/acssynbio.0c00641.
  PUBMED    34075757
REFERENCE   2  (bases 1 to 4686)
  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: "ACS Synth 
            Biol. 2021 Jun 2. doi: 10.1021/acssynbio.0c00641."
COMMENT     Sequence Label: pPAP004
FEATURES             Location/Qualifiers
     source          1..4686
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    63..84
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(99..104,105..122,123..129)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    137..153
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     142..164
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     161..177
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     161..177
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             173..496
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    190..246
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(247..264)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(247..263)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(256..278)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(472..491)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     gene            818..2393
                     /label=hphMX6
                     /note="yeast selectable marker conferring hygromycin 
                     resistance"
     promoter        818..1161
                     /label=TEF promoter
                     /note="Ashbya gossypii TEF promoter"
     CDS             1162..2190
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     CDS             1162..2190
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MGKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGR
                     GYVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDL
                     PETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHW
                     QTVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAM
                     FGDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVD
                     GNFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRA
                     KE"
     terminator      2196..2393
                     /label=TEF terminator
                     /note="Ashbya gossypii TEF terminator"
     promoter        2868..2939
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2940..3008,3009..3800)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3158..3177)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4012..4600
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4501..4520
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgagaccgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
       61 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
      121 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
      181 gattacgcca agcttgcatg cctgcaggtc gactctagag gatccccggg taccgagctc
      241 gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
      301 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
      361 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
      421 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
      481 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
      541 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgacggtct cagctttcga
      601 tttgtatgtg aaatagctga aattcgaaaa tttcattatg gctgtatcta ctttagcgta
      661 ttaggcattt gagcattggc ttgaacaatg cgggctgtag tgtgtcacca aagaaaccat
      721 tcgggttcgg atctggaagt cctcatcacg tgatgccgat ctcgtgtatt ttattttcag
      781 ataacacctg aggactttta cattgtcttc aatacaggac atggaggccc agaataccct
      841 ccttgacagt cttgacgtgc gcagctcagg ggcatgatgt gactgtcgcc cgtacattta
      901 gcccatacat ccccatgtat aatcatttgc atccatacat tttgatggcc gcacggcgcg
      961 aagcaaaaat tacggctcct cgctgcagac gtgcgagcag ggaaacgctc ccctcacaga
     1021 cgcgttgaat tgtccccacg ccgcgcccct gtagagaaat ataaaaggtt aggatttgcc
     1081 actgaggttc ttctttcata tacttccttt taaaatcttg ctaggataca gttctcacat
     1141 cacatccgaa cataaacaac catgggtaaa aagcctgaac tcaccgcgac gtctgtcgag
     1201 aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa
     1261 gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc
     1321 tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc
     1381 ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc
     1441 cgccgtgcac agggtgtcac gttgcaagac ctccctgaaa ccgaactgcc cgctgttctg
     1501 cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg
     1561 ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc
     1621 gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg
     1681 tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg
     1741 cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca
     1801 gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc
     1861 ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg
     1921 catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac
     1981 caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga
     2041 tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga
     2101 agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc
     2161 cccagcactc gtccgagggc aaaggaataa tcagtactga caataaaaag attcttgttt
     2221 tcaagaactt gtcatttgta tagttttttt atattgtagt tgttctattt taatcaaatg
     2281 ttagcgtgat ttatattttt tttcgcctcg acatcatctg cccagatgcg aagttaagtg
     2341 cgcagaaagt aatatcatgc gtcaatcgta tgtgaatgct ggtcgctata ctgggagttc
     2401 aacatctttg gataatatca gaatgagaaa gaacagatac gcagtacgtt ttttggtgag
     2461 ctctttgcac ttctttagtt ctttccatca atatcagttg cttatgcact tatgactaat
     2521 attgatgttt aacttcaata tctttaaact tttgttcttc ccgacgttca ttaagaatac
     2581 taatacactt taataattag tttaatattt gtttctatat aatgacattt aattaaaaaa
     2641 gataaaatat aaaaacatca taataactca ccagaggtta agaacaaaaa aacaaattag
     2701 atatctgcta atccaatata gttaaatcaa tctttccttg gtataatggg tatattacat
     2761 atatttcaag gaccgacact cctaccaaat atctaaaatt taccatatta acataacatg
     2821 tatataaacg tcaaatcata atcagcacta ccgaattttt ctaaatacat tcaaatatgt
     2881 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     2941 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     3001 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     3061 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     3121 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     3181 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     3241 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     3301 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     3361 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     3421 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     3481 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     3541 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     3601 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggttctc
     3661 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     3721 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     3781 cactgattaa gcattggtaa tttcgcgtat ccaacggctg cccaggcagc cgtttttatt
     3841 cctgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     3901 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     3961 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     4021 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     4081 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     4141 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     4201 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     4261 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     4321 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     4381 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     4441 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     4501 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     4561 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     4621 tttacggttc ctgccttttt caatgggtaa taactgatat aattaaattg aacggaagac
     4681 aaaacg
//
