LOCUS       40924_35623        7153 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pPtPuc3, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7153)
  AUTHORS   Karas BJ, Diner RE, Lefebvre SC, Weyman PD.
  TITLE     Designer Diatom episomes Delivered by Bacterial Conjugation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7153)
  AUTHORS   Karas BJ, Diner RE, Lefebvre SC, Weyman PD.
  TITLE     Direct Submission
  JOURNAL   Submitted (03-FEB-2015) Synthetic Biology and Bioenergy Group, J. 
            Craig Venter Institute, 4120 Capricorn Lane, La Jolla, CA 92037, USA
REFERENCE   3  (bases 1 to 7153)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7153)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (03-FEB-2015) Synthetic Biology and Bioenergy Group, J. Craig Venter
            Institute, 4120 Capricorn Lane, La Jolla, CA 92037, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..7153
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(310..678)
                     /codon_start=1
                     /label=traJ
                     /note="oriT-recognizing protein"
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            complement(711..820)
                     /direction=LEFT
                     /label=oriT
                     /note="incP origin of transfer"
     primer_bind     1098..1114
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        1138..1156
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             1860..2231
                     /codon_start=1
                     /label=BleoR
                     /note="antibiotic-binding protein"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     misc_feature    2540..3043
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
     promoter        3044..3230
                     /label=HIS3 promoter
     CDS             3231..3890
                     /codon_start=1
                     /label=HIS3
                     /note="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALGAVRGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHF
                     LESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     primer_bind     complement(3962..3978)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(3986..4002)
                     /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(4010..4040)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4055..4076)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(4364..4952)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             5800..6612
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     promoter        complement(6954..7058)
                     /label=AmpR promoter
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accagatcgt cttgccttgc tcgtcggtga tgtacttaca gctcgaagtg cctcttcttg
      241 atggagcgca tggggacgtg cttggcaatc acgcgcaccc cccggccgtt ttagcggcta
      301 aaaaagtcat ggctctgccc tcgggcggac cacgcccatc atgaccttgc caagctcgtc
      361 ctgcttctct tcgatcttcg ccagcagggc gaggatcgtg gcatcaccga accgcgccgt
      421 gcgcgggtcg tcggtgagcc agagtttcag caggccgccc aggcggccca ggtcgccatt
      481 gatgcgggcc agctcgcgga cgtgctcata gtccacgacg cccgtgattt tgtagccctg
      541 gccgacggcc agcaggtagg ccgacaggct catgccggcc gccgccgcct tttcctcaat
      601 cgctcttcgt tcgtctggaa ggcagtacac cttgataggt gggctgccct tcctggttgg
      661 cttggtttca tcagccatcc gcttgccctc atctgttacg ccggcggtag ccggccagcc
      721 tcgcagagca ggattcccgt tgagcaccgc caggtgcgaa taagggacag tgaagaagga
      781 acacccgctc gcgggtgggc ctacttcacc tatcctgccc ggctgacgcc gttggataca
      841 ccaaggaaag tctacacgaa ccctttggca aaatcctgta tatcgtgcga aaaaggatgg
      901 atataccgaa aaaatcgcta taatgacccc gaagcagggt tatgcagcgg aagatggcca
      961 ttcgccattc aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt
     1021 acgccagctg gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt
     1081 ttcccagtca cgacgttgta aaacgacggc cagtgaccat gattacgcca agctcgaaat
     1141 taaccctcac taaagggaac aaaagctggt acctaacagg attagtgcaa ttcgagttga
     1201 atcactggga aaaacattgt cttctttttt atattatcat ttgcattagt gctgcagtcg
     1261 tagatacttg ttggttgaaa gacatcagct gggagggact ggactagcgt ttggtaagga
     1321 gacatacctg ttaacgttgg ttgcaaaatt ccatttcgcg atttatgtta tctgtaaatc
     1381 ctgatttgtc tggaattctt gatacttccg tttttttaga ggccaatgat tagcatcggc
     1441 gattctcaaa atagcatttt cgacatgcgg tgctgatttc ataaacatag acaacgcttt
     1501 tacatgtaaa agtaacttgc ggacttggaa cagtgctctg tttttggtgt gaacgtaact
     1561 cagcaatatt tctgtgctag caaggttttt tatgatcgac cgaagatctc aaaactccgg
     1621 gtctttcaac tgtctgacta gaccatgttc gtaacgtcgg acagcagctt tcgttgtact
     1681 ggtagaattt ctacgtgcga agcacgtgta ggcaggttga acgacgatcc ctgccgatgg
     1741 atggattggc acgcggcgga acgctttcgt gatctacacc acctggatct tcacatatct
     1801 tcgaaatcga aaaattaacc aagtcgacgg tatcgataat attctagctg agggtaccca
     1861 tggccaagtt gaccagtgcc gttccggtgc tcaccgcgcg cgacgtcgcc ggagcggtcg
     1921 agttctggac cgaccggctc gggttctccc gggacttcgt ggaggacgac ttcgccggtg
     1981 tggtccggga cgacgtgacc ctgttcatca gcgcggtcca ggaccaggtg gtgccggaca
     2041 acaccctggc ctgggtgtgg gtgcgcggcc tggacgagct gtacgccgag tggtcggagg
     2101 tcgtgtccac gaacttccgg gacgcctccg ggccggccat gaccgagatc ggcgagcagc
     2161 cgtgggggcg ggagttcgcc ctgcgcgacc cggccggcaa ctgcgtgcac ttcgtggccg
     2221 aggagcagga ctgaccgacg ccgaccaaca ccgccggtcc gacgcggccc gacgggtccg
     2281 aggcctcgga gatctgggcc catgcggccg caacaactac ctcgactttg gctgggacac
     2341 tttcagtgag gacaagaagc ttcagaagcg tgctatcgaa ctcaaccagg gacgtgcggc
     2401 acaaatgggc atccttgctc tcatggtgca cgaacagttg ggagtctcta tccttcctta
     2461 aaaatttaat tttcattagt tgcagtcact ccgctttggt ttcacagtca ggaataacac
     2521 tagctcgtct tcagcgagca tcacgtgcta taaaaataat tataatttaa attttttaat
     2581 ataaatatat aaattaaaaa tagaaagtaa aaaaagaaat taaagaaaaa atagtttttg
     2641 ttttccgaag atgtaaaaga ctctaggggg atcgccaaca aatactacct tttaccttgc
     2701 tcttcctgct ctcaggtatt aatgccgaat tgtttcatct tgtctgtgta gaagaccaca
     2761 cacgaaaatc ctgtgatttt acattttact tatcgttaat cgaatgtata tctatttaat
     2821 ctgcttttct tgtctaataa atatatatgt aaagtacgct ttttgttgaa attttttaaa
     2881 cctttgttta tttttttttc ttcattccgt aactcttcta ccttctttat ttactttcta
     2941 aaatccaaat acaaaacata aaaataaata aacacagagt aaattcccaa attattccat
     3001 cattaaaaga tacgaggcgc gtgtaagtta caggcaagcg atcctagtac actctatatt
     3061 tttttatgcc tcggtaatga ttttcatttt ttttttccac ctagcggatg actctttttt
     3121 tttcttagcg attggcatta tcacataatg aattatacat tatataaagt aatgtgattt
     3181 cttcgaagaa tatactaaaa aatgagcagg caagataaac gaaggcaaag atgacagagc
     3241 agaaagccct agtaaagcgt attacaaatg aaaccaagat tcagattgcg atctctttaa
     3301 agggtggtcc cctagcgata gagcactcga tcttcccaga aaaagaggca gaagcagtag
     3361 cagaacaggc cacacaatcg caagtgatta acgtccacac aggtataggg tttctggacc
     3421 atatgataca tgctctggcc aagcattccg gctggtcgct aatcgttgag tgcattggtg
     3481 acttacacat agacgaccat cacaccactg aagactgcgg gattgctctc ggtcaagctt
     3541 ttaaagaggc cctaggggcc gtgcgtggag taaaaaggtt tggatcagga tttgcgcctt
     3601 tggatgaggc actttccaga gcggtggtag atctttcgaa caggccgtac gcagttgtcg
     3661 aacttggttt gcaaagggag aaagtaggag atctctcttg cgagatgatc ccgcattttc
     3721 ttgaaagctt tgcagaggct agcagaatta ccctccacgt tgattgtctg cgaggcaaga
     3781 atgatcatca ccgtagtgag agtgcgttca aggctcttgc ggttgccata agagaagcca
     3841 cctcgcccaa tggtaccaac gatgttccct ccaccaaagg tgttcttatg tagttttaca
     3901 caggagtctg gacttgacct ctagagtcga cctgcaggca tgcaagcttg gcgtaatcat
     3961 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     4021 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     4081 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     4141 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     4201 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4261 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4321 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4381 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4441 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4501 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4561 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4621 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4681 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4741 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4801 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4861 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4921 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4981 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     5041 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     5101 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     5161 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     5221 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5281 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5341 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5401 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5461 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5521 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5581 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5641 tgccatccgt aagatgcttt tctgtgactg gtgagtcgat ttattcaaca aagccacgtt
     5701 gtgtctcaaa atctctgatg ttacattgca caagataaaa atatatcatc atgaacaata
     5761 aaactgtctg cttacataaa cagtaataca aggggtgtta tgagccatat tcaacgggaa
     5821 acgtcttgct cgaggccgcg attaaattcc aacatggatg ctgatttata tgggtataaa
     5881 tgggctcgcg ataatgtcgg gcaatcaggt gcgacaatct atcgattgta tgggaagccc
     5941 gatgcgccag agttgtttct gaaacatggc aaaggtagcg ttgccaatga tgttacagat
     6001 gagatggtca gactaaactg gctgacggaa tttatgcctc ttccgaccat caagcatttt
     6061 atccgtactc ctgatgatgc atggttactc accactgcga tccccgggaa aacagcattc
     6121 caggtattag aagaatatcc tgattcaggt gaaaatattg ttgatgcgct ggcagtgttc
     6181 ctgcgccggt tgcattcgat tcctgtttgt aattgtcctt ttaacagcga tcgcgtattt
     6241 cgtctcgctc aggcgcaatc acgaatgaat aacggtttgg ttgatgcgag tgattttgat
     6301 gacgagcgta atggctggcc tgttgaacaa gtctggaaag aaatgcataa gcttttgcca
     6361 ttctcaccgg attcagtcgt cactcatggt gatttctcac ttgataacct tatttttgac
     6421 gaggggaaat taataggttg tattgatgtt ggacgagtcg gaatcgcaga ccgataccag
     6481 gatcttgcca tcctatggaa ctgcctcggt gagttttctc cttcattaca gaaacggctt
     6541 tttcaaaaat atggtattga taatcctgat atgaataaat tgcagtttca tttgatgctc
     6601 gatgagtttt tctaatcaga attggttaat tggttgtaac actggcactc aaccaagtca
     6661 ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat
     6721 accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga
     6781 aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc
     6841 aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg
     6901 caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc
     6961 ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt
     7021 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
     7081 cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg
     7141 aggccctttc gtc
//
