LOCUS       62056_18515        7973 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 7973)
  AUTHORS   .
  TITLE     Direct Submission
FEATURES             Location/Qualifiers
     source          1..7973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        2..940
                     /label=AOX1 promoter
                     /note="inducible promoter, regulated by methanol"
     CDS             947..1201
                     /codon_start=1
                     /label=alpha-factor secretion signal
                     /note="N-terminal secretion signal from S. cerevisiae 
                     alpha-factor"
                     /translation="MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLE
                     GDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR"
     terminator      1254..1503
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     promoter        1523..1604
                     /label=PpADE2 LC promoter
                     /note="strong 82-bp promoter fragment that requires only 
                     low-copy integration for efficient expression"
     CDS             1605..3293
                     /codon_start=1
                     /label=PpADE2
                     /note="phosphoribosylaminoimidazole carboxylase from Pichia
                     pastoris"
                     /translation="MDSQVIGILGGGQLGRMIVEAASRLNIKTVILDDGFSPAKHINAA
                     QDHIDGSFKDEEAIAKLAAKCDVLTVEIEHVNTDALKRVQDRTGIKIYPLPETIELIKD
                     KYLQKEHLIKHNISVTKSQGIESNEKALLLFGEENGFPYLLKSRTMAYDGRGNFVVESK
                     EDISKALEFLKDRPLYAEKFAPFVKELAVMVVRSLEGEVFSYPTVETVHKDNICHIVYA
                     PARVNDTIQKKAQILAENTVKTFPGAGIFGVEMFLLSDGELLVNEIAPRPHNSGHYTID
                     ACVTSQFEAHVRAITGLPMPLDFTKLSTSNTNAIMLNVLGAEKSHGELEFCRRALETPG
                     ASVYLYGKTTRLARKMGHINIIGSSMLEAEQKLEYILEESTHLPSSTVSADTKPLVGVI
                     MGSDSDLPVISKGCDILKQFGVPFEVTIVSAHRTPQRMTRYAFEAASRGIKAIIAGAGG
                     AAHLPGMVAAMTPLPVIGVPVKGSTLDGVDSLHSIVQMPRGVPVATVAINNATNAALLA
                     IRILGTIDHKWQKEMSKYMNAMETEVLGKASNLESEGYESYLKNRL"
     promoter        3523..3672
                     /label=PpTRP2 promoter
                     /note="promoter for Pichia pastoris TRP2"
     CDS             3673..5286
                     /codon_start=1
                     /label=PpTRP2
                     /note="anthranilate synthase from Pichia pastoris"
                     /translation="MSVSDSHLATDYFGTQLTKQIHPSAESFLDIVKNDRQSHSPETVP
                     NLYPIYKFIQSDFLTPHLAYIKLTNSSETPSPSFLLESAKNGDTVDRYTFMGHSPRKII
                     KTGPLEGAEVDPLVLLEKELKGTRQAQLPGIPRLSGGAIGYISYDCIKYFEPKTERKLK
                     DVLQLPEAALMLFDTIVAFDNVYQRFQVIGNVSLSVDDSDEAILEKYYKTREEVEKISK
                     VVFDNKTVPYYEQKDIIQGQTFTSNIGQEGYENHVRKLKEHILKGDIFQAVPSQRVARP
                     TSLHPFNIYRHLRTVNPSPYMFYIDYLDFQVVGASPELLVKSDNNNKIITHPIAGTLPR
                     GKTIEEDDNYAKQLKSSLKDRAEHVMLVDLARNDINRVCEPTSTTVDRLLTVERFSHVM
                     HLVSEVSGTLRPNKTRFDAFRSIFPAGTVSGAPKVRAMQLIGELEGEKRGVYAGAVGHW
                     SYDGKSMDTCIALRTMVVKDGVAYLQAGGGIVYDSDPYDEYIETMNKMRSNNNTILEAE
                     KIWTDRLARDENQSESEENDQ"
     primer_bind     complement(5351..5367)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(5375..5391)
                     /label=lac operator
                     /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)."
     promoter        complement(5399..5429)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(5444..5465)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5753..6341)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(6515..7372)
                     /codon_start=1
                     /label=AmpR
                     /note="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     promoter        complement(7373..7477)
                     /label=AmpR promoter
     primer_bind     7951..7967
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
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 aaagtcggca 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 attcgaaacg gaattcatga gatttccttc
      961 aatttttact gctgttttat tcgcagcatc ctccgcatta gctgctccag tcaacactac
     1021 aacagaagat gaaacggcac aaattccggc tgaagctgtc atcggttact cagatttaga
     1081 aggggatttc gatgttgctg ttttgccatt ttccaacagc acaaataacg ggttattgtt
     1141 tataaatact actattgcca gcattgctgc taaagaagaa ggggtatctc tcgagaaaag
     1201 gcctcaggta ccggccggcc atttaaatac aggccccttt tcctttgtcg atatcatgta
     1261 attagttatg tcacgcttac attcacgccc tcctcccaca tccgctctaa ccgaaaagga
     1321 aggagttaga caacctgaag tctaggtccc tatttatttt ttttaatagt tatgttagta
     1381 ttaagaacgt tatttatatt tcaaattttt cttttttttc tgtacaaacg cgtgtacgca
     1441 tgtaacatta tactgaaaac cttgcttgag aaggttttgg gacgctcgaa ggctttaatt
     1501 tgcaagctgg atccgcggcc gcgacttgac tcttcactag cctatgcaaa taaggttacc
     1561 ttttccaaga atcgtagaaa cgattaaaaa acttccaaac tctcatggat tctcaggtaa
     1621 taggtattct aggaggaggc cagctaggcc gaatgattgt tgaggccgct agcaggctca
     1681 atatcaagac cgtgattctt gatgatggtt tttcacctgc taagcacatt aatgctgcgc
     1741 aagaccacat cgacggatca ttcaaagatg aggaggctat cgccaagtta gctgccaaat
     1801 gtgatgttct cactgtagag attgagcatg tcaacacaga tgctctaaag agagttcaag
     1861 acagaactgg aatcaagata tatcctttac cagagacaat cgaactaatc aaggataagt
     1921 acttgcaaaa ggaacatttg atcaagcaca acatttcggt gacaaagtct cagggtatag
     1981 aatctaatga aaaggcgctg cttttgtttg gagaagagaa tggatttcca tatctgttga
     2041 agtcccggac tatggcttat gatggaagag gcaattttgt agtggagtct aaagaggaca
     2101 tcagtaaggc attagagttc ttgaaagatc gtccattgta tgccgagaag tttgctcctt
     2161 ttgttaaaga attagcggta atggttgtga gatcactgga aggcgaagta ttctcctacc
     2221 caaccgtaga aactgtgcac aaggacaata tctgtcatat tgtgtatgct ccggccagag
     2281 ttaatgacac catccaaaag aaagctcaaa tattagctga aaacactgtg aagactttcc
     2341 caggcgctgg aatcttcgga gttgagatgt tcctattgtc tgatggagaa cttcttgtaa
     2401 atgagattgc tccaaggccc cacaattctg gtcactatac aatcgatgca tgtgtaacat
     2461 ctcagttcga agcacatgta agagccataa ctggtctgcc aatgccacta gatttcacca
     2521 aactatctac ttccaacacc aacgctatta tgctcaatgt tttgggtgct gaaaaatctc
     2581 acggggaatt agagttttgt agaagagcct tagaaacacc cggtgcttct gtatatctgt
     2641 acggaaagac cacccgattg gctcgtaaga tgggtcatat caacataata ggatcttcca
     2701 tgttggaagc agaacaaaag ttagagtaca ttctagaaga atcaacccac ttaccatcca
     2761 gtactgtatc agctgacact aaaccgttgg ttggagttat catgggttca gactctgatc
     2821 tacctgtgat ttcgaaaggt tgcgatattt taaaacagtt tggtgttcca ttcgaagtta
     2881 ctattgtctc tgctcataga acaccacaga gaatgaccag atatgccttt gaagccgcta
     2941 gtagaggtat caaggctatc attgcaggtg ctggtggtgc tgctcatctt ccaggaatgg
     3001 ttgctgccat gactccgttg ccagtcattg gtgttcctgt caagggctct acgttggatg
     3061 gtgtagactc gctacactcg attgtccaaa tgcctagagg tgttcctgtg gctacggttg
     3121 ctatcaacaa cgccaccaat gccgctctgt tggccatcag gattttaggt acaattgacc
     3181 acaaatggca aaaggaaatg tccaagtata tgaatgcaat ggagaccgaa gtgttgggga
     3241 aggcatccaa cttggaatct gaagggtatg aatcctattt gaagaatcgt ctttgaattt
     3301 agtattgttt tttaatagat gtatatataa tagtacacgt aacttatcta ttccattcat
     3361 aattttattt taaaggttcg gtagaaattt gtcctccaaa aagttggtta gagcctggca
     3421 gttttgatag gcattattat agattgggta atatttaccc tgcacctgga ggaactttgc
     3481 aaagagcctc atgtgcggcg cgccaggcca taatggccaa acggtttctc aattactata
     3541 tactactaac catttacctg tagcgtattt cttttccctc ttcgcgaaag ctcaagggca
     3601 tcttcttgac tcatgaaaaa tatctggatt tcttctgaca gatcatcacc cttgagccca
     3661 actctctagc ctatgagtgt aagtgatagt catcttgcaa cagattattt tggaacgcaa
     3721 ctaacaaagc agatacaccc ttcagcagaa tcctttctgg atattgtgaa gaatgatcgc
     3781 caaagtcaca gtcctgagac agttcctaat ctttacccca tttacaagtt catccaatca
     3841 gacttcttaa cgcctcatct ggcttatatc aagcttacca acagttcaga aactcccagt
     3901 ccaagtttct tgcttgaaag tgcgaagaat ggtgacaccg ttgacaggta cacctttatg
     3961 ggacattccc ccagaaaaat aatcaagact gggcctttag agggtgctga agttgacccc
     4021 ttggtgcttc tggaaaaaga actgaagggc accagacaag cgcaacttcc tggtattcct
     4081 cgtctaagtg gtggtgccat aggatacatc tcgtacgatt gtattaagta ctttgaacca
     4141 aaaactgaaa gaaaactgaa agatgttttg caacttccgg aagcagcttt gatgttgttc
     4201 gacacgatcg tggcttttga caatgtttat caaagattcc aggtaattgg aaacgtttct
     4261 ctatccgttg atgactcgga cgaagctatt cttgagaaat attataagac aagagaagaa
     4321 gtggaaaaga tcagtaaagt ggtatttgac aataaaactg ttccctacta tgaacagaaa
     4381 gatattattc aaggccaaac gttcacctct aatattggtc aggaagggta tgaaaaccat
     4441 gttcgcaagc tgaaagaaca tattctgaaa ggagacatct tccaagctgt tccctctcaa
     4501 agggtagcca ggccgacctc attgcaccct ttcaacatct atcgtcattt gagaactgtc
     4561 aatccttctc catacatgtt ctatattgac tatctagact tccaagttgt tggtgcttca
     4621 cctgaattac tagttaaatc cgacaacaac aacaaaatca tcacacatcc tattgctgga
     4681 actcttccca gaggtaaaac tatcgaagag gacgacaatt atgctaagca attgaagtcg
     4741 tctttgaaag acagggccga gcacgtcatg ctggtagatt tggccagaaa tgatattaac
     4801 cgtgtgtgtg agcccaccag taccacggtt gatcgtttat tgactgtgga gagattttct
     4861 catgtgatgc atcttgtgtc agaagtcagt ggaacattga gaccaaacaa gactcgcttc
     4921 gatgctttca gatccatttt cccagcagga accgtctccg gtgctccgaa ggtaagagca
     4981 atgcaactca taggagaatt ggaaggagaa aagagaggtg tttatgcggg ggccgtagga
     5041 cactggtcgt acgatggaaa atcgatggac acatgtattg ccttaagaac aatggtcgtc
     5101 aaggacggtg tcgcttacct tcaagccgga ggtggaattg tctacgattc tgacccctat
     5161 gacgagtaca tcgaaaccat gaacaaaatg agatccaaca ataacaccat cttggaggct
     5221 gagaaaatct ggaccgatag gttggccaga gacgagaatc aaagtgaatc cgaagaaaac
     5281 gatcaatgaa cggaggacgt aagtaggaat ttatggtttg gccataatgg cctagcttgg
     5341 cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca
     5401 acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca
     5461 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5521 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     5581 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     5641 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     5701 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     5761 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     5821 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     5881 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     5941 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6001 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     6061 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     6121 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     6181 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     6241 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     6301 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     6361 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     6421 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     6481 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     6541 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     6601 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     6661 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     6721 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     6781 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     6841 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     6901 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6961 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7021 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     7081 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     7141 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     7201 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     7261 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     7321 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     7381 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     7441 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     7501 ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg cgtatcacga
     7561 ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac atgcagctcc
     7621 cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg
     7681 cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca gagcagattg
     7741 tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg agaaaatacc
     7801 gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga tcggtgcggg
     7861 cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga ttaagttggg
     7921 taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa ttg
//
