LOCUS       62056_18470        9972 bp DNA     circular SYN 01-JAN-1980
DEFINITION  synthetic circular DNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9972)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..9972
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..937
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             949..1215
                     /codon_start=1
                     /label=alpha-factor secretion signal
                     /note="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKREAEA"
     CDS             1924..1941
                     /codon_start=1
                     /label=6xHis
                     /note="6xHis affinity tag"
                     /translation="HHHHHH"
     terminator      2017..2263
                     /label=AOX1 terminator
                     /note="transcription terminator for AOX1"
     CDS             complement(5627..6439)
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     misc_feature    6818..7574
                     /label=AOX1 3' fragment
                     /note="region downstream of Pichia pastoris AOX1 gene"
     misc_feature    7717..7857
                     /label=bom
                     /note="basis of mobility region from pBR322"
     rep_origin      complement(8043..8631)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(8805..9662)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(9663..9767)
                     /label=AmpR promoter
ORIGIN
        1 agatctaaca tccaaagacg aaaggttgaa tgaaaccttt ttgccatccg acatccacag
       61 gtccattctc acacataagt gccaaacgca acaggagggg atacactagc agcagaccgt
      121 tgcaaacgca ggacctccac tcctcttctc ctcaacaccc acttttgcca tcgaaaaacc
      181 agcccagtta ttgggcttga ttggagctcg ctcattccaa ttccttctat taggctacta
      241 acaccatgac tttattagcc tgtctatcct ggcccccctg gcgaggttca tgtttgttta
      301 tttccgaatg caacaagctc cgcattacac ccgaacatca ctccagatga gggctttctg
      361 agtgtggggt caaatagttt catgttcccc aaatggccca aaactgacag tttaaacgct
      421 gtcttggaac ctaatatgac aaaagcgtga tctcatccaa gatgaactaa gtttggttcg
      481 ttgaaatgct aacggccagt tggtcaaaaa gaaacttcca aaagtcgcca taccgtttgt
      541 cttgtttggt attgattgac gaatgctcaa aaataatctc attaatgctt agcgcagtct
      601 ctctatcgct tctgaacccc ggtgcacctg tgccgaaacg caaatgggga aacacccgct
      661 ttttggatga ttatgcattg tctccacatt gtatgcttcc aagattctgg tgggaatact
      721 gctgatagcc taacgttcat gatcaaaatt taactgttct aacccctact tgacagcaat
      781 atataaacag aaggaagctg ccctgtctta aacctttttt tttatcatca ttattagctt
      841 actttcataa ttgcgactgg ttccaattga caagcttttg attttaacga cttttaacga
      901 caacttgaga agatcaaaaa acaactaatt attcgaagga tccaaacgat gagatttcct
      961 tcaattttta ctgcagtttt attcgcagca tcctccgcat tagctgctcc agtcaacact
     1021 acaacagaag atgaaacggc acaaattccg gctgaagctg tcatcggtta ctcagattta
     1081 gaaggggatt tcgatgttgc tgttttgcca ttttccaaca gcacaaataa cgggttattg
     1141 tttataaata ctactattgc cagcattgct gctaaagaag aaggggtatc tctcgagaaa
     1201 agagaggctg aagctgcgac gggactcgac gacccggcga agaaggagat cgccatgcaa
     1261 ctggtgtcga gcgccgagaa ctcctcgctc gactggaagg cccagtaccg gtacatcgag
     1321 gacatcggtg acggtcgcgg ctacaccgcg ggcatcatcg gcttctgttc cggcaccggc
     1381 gacatgctcg acctggtgga gctgtacggc gagcgcagcc ccgggaacgt cctcgcgccc
     1441 tatctgccgg cgctgcgccg ggtggacggc tccgactcgc acgagggcct cgaccccggc
     1501 ttcccggacg actggcgccg ggccgccgac caggacccgc agttccgccg ggcccaggac
     1561 gacgagcggg accgcgtgta cttcgacccg gccgtacggc gcggcaagga ggacgggctg
     1621 cggacgctgg ggcagttcgc gtactacgac gcgatggtga tgcacggcga cggcggcggc
     1681 ctcggcttcg gcagtatccg cgagcgcgcc ctcggccggg cccgcccgcc ggcccagggc
     1741 ggtgacgagg tcgcctacct gcacgccttc ctcgacgagc gggtgtgggc catgaagcag
     1801 gagcaggcgc acagcgacac cagccgggtc gacacggcgc agcgcgtctt cctgaacgag
     1861 ggcaacctgg acctcgagcc gccgctggac tggcacgtgt acggggacgc ctaccacatc
     1921 ggccatcatc accatcacca ttaattcgcc ttagacatga ctgttcctca gttcaagttg
     1981 ggcacttacg agaagaccgg tcttgctaga ttctaatcaa gaggatgtca gaatgccatt
     2041 tgcctgagag atgcaggctt catttttgat acttttttat ttgtaaccta tatagtatag
     2101 gatttttttt gtcattttgt ttcttctcgt acgagcttgc tcctgatcag cctatctcgc
     2161 agctgatgaa tatcttgtgg taggggtttg ggaaaatcat tcgagtttga tgtttttctt
     2221 ggtatttccc actcctcttc agagtacaga agattaagtg agaagttcgt ttgtgcaagc
     2281 ttatcgataa gctttaatgc ggtagtttat cacagttaaa ttgctaacgc agtcaggcac
     2341 cgtgtatgaa atctaacaat gcgctcatcg tcatcctcgg caccgtcacc ctggatgctg
     2401 taggcatagg cttggttatg ccggtactgc cgggcctctt gcgggatatc gtccattccg
     2461 acagcatcgc cagtcactat ggcgtgctgc tagcgctata tgcgttgatg caatttctat
     2521 gcgcacccgt tctcggagca ctgtccgacc gctttggccg ccgcccagtc ctgctcgctt
     2581 cgctacttgg agccactatc gactacgcga tcatggcgac cacacccgtc ctgtggatct
     2641 atcgaatcta aatgtaagtt aaaatctcta aataattaaa taagtcccag tttctccata
     2701 cgaaccttaa cagcattgcg gtgagcatct agaccttcaa cagcagccag atccatcact
     2761 gcttggccaa tatgtttcag tccctcagga gttacgtctt gtgaagtgat gaacttctgg
     2821 aaggttgcag tgttaactcc gctgtattga cgggcatatc cgtacgttgg caaagtgtgg
     2881 ttggtaccgg aggagtaatc tccacaactc tctggagagt aggcaccaac aaacacagat
     2941 ccagcgtgtt gtacttgatc aacataagaa gaagcattct cgatttgcag gatcaagtgt
     3001 tcaggagcgt actgattgga catttccaaa gcctgctcgt aggttgcaac cgatagggtt
     3061 gtagagtgtg caatacactt gcgtacaatt tcaacccttg gcaactgcac agcttggttg
     3121 tgaacagcat cttcaattct ggcaagctcc ttgtctgtca tatcgacagc caacagaatc
     3181 acctgggaat caataccatg ttcagcttga gacagaaggt ctgaggcaac gaaatctgga
     3241 tcagcgtatt tatcagcaat aactagaact tcagaaggcc cagcaggcat gtcaatacta
     3301 cacagggctg atgtgtcatt ttgaaccatc atcttggcag cagtaacgaa ctggtttcct
     3361 ggaccaaata ttttgtcaca cttaggaaca gtttctgttc cgtaagccat agcagctact
     3421 gcctgggcgc ctcctgctag cacgatacac ttagcaccaa ccttgtgggc aacgtagatg
     3481 acttctgggg taagggtacc atccttctta ggtggagatg caaaaacaat ttctttgcaa
     3541 ccagcaactt tggcaggaac acccagcatc agggaagtgg aaggcagaat tgcggttcca
     3601 ccaggaatat agaggccaac tttctcaata ggtcttgcaa aacgagagca gactacacca
     3661 gggcaagtct caacttgcaa cgtctccgtt agttgagctt catggaattt cctgacgtta
     3721 tctatagaga gatcaatggc tctcttaacg ttatctggca attgcataag ttcctctggg
     3781 aaaggagctt ctaacacagg tgtcttcaaa gcgactccat caaacttggc agttagttct
     3841 aaaagggctt tgtcaccatt ttgacgaaca ttgtcgacaa ttggtttgac taattccata
     3901 atctgttccg ttttctggat aggacgacga agggcatctt caatttcttg tgaggaggcc
     3961 ttagaaacgt caattttgca caattcaata cgaccttcag aagggacttc tttaggtttg
     4021 gattcttctt taggttgttc cttggtgtat cctggcttgg catctccttt ccttctagtg
     4081 acctttaggg acttcatatc caggtttctc tccacctcgt ccaacgtcac accgtacttg
     4141 gcacatctaa ctaatgcaaa ataaaataag tcagcacatt cccaggctat atcttccttg
     4201 gatttagctt ctgcaagttc atcagcttcc tccctaattt tagcgttcaa caaaacttcg
     4261 tcgtcaaata accgtttggt ataagaacct tctggagcat tgctcttacg atcccacaag
     4321 gtggcttcca tggctctaag accctttgat tggccaaaac aggaagtgcg ttccaagtga
     4381 cagaaaccaa cacctgtttg ttcaaccaca aatttcaagc agtctccatc acaatccaat
     4441 tcgataccca gcaacttttg agttgctcca gatgtagcac ctttatacca caaaccgtga
     4501 cgacgagatt ggtagactcc agtttgtgtc cttatagcct ccggaataga ctttttggac
     4561 gagtacacca ggcccaacga gtaattagaa gagtcagcca ccaaagtagt gaatagacca
     4621 tcggggcggt cagtagtcaa agacgccaac aaaatttcac tgacagggaa ctttttgaca
     4681 tcttcagaaa gttcgtattc agtagtcaat tgccgagcat caataatggg gattatacca
     4741 gaagcaacag tggaagtcac atctaccaac tttgcggtct cagaaaaagc ataaacagtt
     4801 ctactaccgc cattagtgaa acttttcaaa tcgcccagtg gagaagaaaa aggcacagcg
     4861 atactagcat tagcgggcaa ggatgcaact ttatcaacca gggtcctata gataacccta
     4921 gcgcctggga tcatcctttg gacaactctt tctgccaaat ctaggtccaa aatcacttca
     4981 ttgataccat tattgtacaa cttgagcaag ttgtcgatca gctcctcaaa ttggtcctct
     5041 gtaacggatg actcaacttg cacattaact tgaagctcag tcgattgagt gaacttgatc
     5101 aggttgtgca gctggtcagc agcataggga aacacggctt ttcctaccaa actcaaggaa
     5161 ttatcaaact ctgcaacact tgcgtatgca ggtagcaagg gaaatgtcat acttgaagtc
     5221 ggacagtgag tgtagtcttg agaaattctg aagccgtatt tttattatca gtgagtcagt
     5281 catcaggaga tcctctacgc cggacgcatc gtggccgacc tgcagggggg gggggggcgc
     5341 tgaggtctgc ctcgtgaaga aggtgttgct gactcatacc aggcctgaat cgccccatca
     5401 tccagccaga aagtgaggga gccacggttg atgagagctt tgttgtaggt ggaccagttg
     5461 gtgattttga acttttgctt tgccacggaa cggtctgcgt tgtcgggaag atgcgtgatc
     5521 tgatccttca actcagcaaa agttcgattt attcaacaaa gccgccgtcc cgtcaagtca
     5581 gcgtaatgct ctgccagtgt tacaaccaat taaccaattc tgattagaaa aactcatcga
     5641 gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa
     5701 gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct
     5761 ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt
     5821 caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg
     5881 gcaaaagctt atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat
     5941 caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa
     6001 atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga
     6061 acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga
     6121 atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa
     6181 aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat
     6241 ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg
     6301 gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt
     6361 tatacccata taaatcagca tccatgttgg aatttaatcg cggcctcgag caagacgttt
     6421 cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta
     6481 ttgttcatga tgatatattt ttatcttgtg caatgtaaca tcagagattt tgagacacaa
     6541 cgtggctttc cccccccccc ctgcaggtcg gcatcaccgg cgccacaggt gcggttgctg
     6601 gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc gggctcatga
     6661 gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg ttgggcgcca
     6721 tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac ctactactgg
     6781 gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agtatctatg attggaagta
     6841 tgggaatggt gatacccgca ttcttcagtg tcttgaggtc tcctatcaga ttatgcccaa
     6901 ctaaagcaac cggaggagga gatttcatgg taaatttctc tgacttttgg tcatcagtag
     6961 actcgaactg tgagactatc tcggttatga cagcagaaat gtccttcttg gagacagtaa
     7021 atgaagtccc accaataaag aaatccttgt tatcaggaac aaacttcttg tttcgaactt
     7081 tttcggtgcc ttgaactata aaatgtagag tggatatgtc gggtaggaat ggagcgggca
     7141 aatgcttacc ttctggacct tcaagaggta tgtagggttt gtagatactg atgccaactt
     7201 cagtgacaac gttgctattt cgttcaaacc attccgaatc cagagaaatc aaagttgttt
     7261 gtctactatt gatccaagcc agtgcggtct tgaaactgac aatagtgtgc tcgtgttttg
     7321 aggtcatctt tgtatgaata aatctagtct ttgatctaaa taatcttgac gagccaaggc
     7381 gataaatacc caaatctaaa actcttttaa aacgttaaaa ggacaagtat gtctgcctgt
     7441 attaaacccc aaatcagctc gtagtctgat cctcatcaac ttgaggggca ctatcttgtt
     7501 ttagagaaat ttgcggagat gcgatatcga gaaaaaggta cgctgatttt aaacgtgaaa
     7561 tttatctcaa gatctctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
     7621 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
     7681 tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag
     7741 cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg
     7801 caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc
     7861 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     7921 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     7981 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     8041 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     8101 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     8161 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     8221 agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     8281 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     8341 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     8401 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     8461 cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt
     8521 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     8581 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     8641 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     8701 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     8761 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     8821 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     8881 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     8941 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     9001 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     9061 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctgca
     9121 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     9181 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     9241 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     9301 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     9361 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaaca
     9421 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     9481 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     9541 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     9601 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     9661 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     9721 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     9781 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     9841 cgtatcacga ggccctttcg tcttcaagaa ttaattctca tgtttgacag cttatcatcg
     9901 ataagctgac tcatgttggt attgtgaaat agacgcagat cgggaacact gaaaaataac
     9961 agttattatt cg
//
