LOCUS       40924_43823        7796 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Cloning vector pTpPuc3, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7796)
  AUTHORS   Karas BJ, Diner RE, Lefebvre SC, Weyman PD.
  TITLE     Designer Diatom episomes Delivered by Bacterial Conjugation
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7796)
  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 7796)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 7796)
  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            "Unpublished"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"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..7796
                     /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"
     CDS             2096..2662
                     /codon_start=1
                     /label=NrsR
                     /note="nourseothricin acetyltransferase"
                     /translation="MTTLDDTAYRYRTSVPGDAEAIEALDGSFTTDTVFRVTATGDGFT
                     LREVPVDPPLTKVFPDDESDDESDAGEDGDPDSRTFVAYGDDGDLAGFVVVSYSGWNRR
                     LTVEDIEVAPEHRGHGVGRALMGLATEFARERGAGHLWLEVTNVNAPAIHAYRRMGFTL
                     CGLDTALYDGTASDGEQALYMSMPCP"
     misc_feature    3185..3688
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
     promoter        3689..3876
                     /label=HIS3 promoter
     CDS             3877..4533
                     /codon_start=1
                     /label=HIS3
                     /note="imidazoleglycerol-phosphate dehydratase, required
                     for histidine biosynthesis"
                     /translation="MTEQKALVKRITNETKIQIAISLKGGPLAIEHSIFPEKEAEAVAE
                     QATQSQVINVHTGIGFLDHMIHALAKHSGWSLIVECIGDLHIDDHHTTEDCGIALGQAF
                     KEALLARGVKRFGSGFAPLDEALSRAVVDLSNRPYAVVELGLQREKVGDLSCEMIPHFL
                     ESFAEASRITLHVDCLRGKNDHHRSESAFKALAVAIREATSPNGTNDVPSTKGVLM"
     primer_bind     complement(4605..4621)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(4629..4645)
                     /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(4653..4683)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    complement(4698..4719)
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     rep_origin      complement(5007..5595)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             6443..7255
                     /codon_start=1
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     promoter        complement(7597..7701)
                     /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 cagtgagctt gcgctttttc cgagaactcc
     1141 ccataagtca acggctccaa tcaagaatgt atccgacaac ggcgagcata gcaacacgtc
     1201 cgtctttgga gtagaatcat catgttgtgg atgaatacac agatgaatga cattaaaagc
     1261 atgaacatgt tagagagtag gaggtagaga ttgatatggt agcattgcga tgtttgtttt
     1321 tggtcagcat atgatgagtg gataccaata tgatgaaagt tgaatctcgc gtttgagctc
     1381 agcggtacgt tattgatcga aagtagcctg atcaaaatcc ttggagagta caagaggatc
     1441 aaagaatcca gtgggggcga taactccaag ctcgttctca aagaggcaat ggaggtagaa
     1501 actcatccca gttgagaaga agtgaaggca gtggcggtgg cgaaagcaga ggcaacgagg
     1561 acagacttcc tgtgggttga tgcaacgaat atttccagaa ggagaagttt agagagttga
     1621 accgctacct acaatgacaa agtatcgtat cgattttgat gttggttggt tatgaattca
     1681 aactgtaagt tggattgtga gaagatcaga agttgaacga acacatcttt ccgatcattc
     1741 acctccacac tgcaacaaca cggtacttct tccgcggcag gtctctgtcg ccattctctt
     1801 gtcctgttgt tggctgtgag acgaggaaag caacgacaag tttcacaaaa gggagttcct
     1861 ttaacgagat atgtttttta taaagagtcc caatagaaag acaaattgat tcctccgtgc
     1921 aaacgcgcaa ataaacacca cgtccattat atccatatct ttcagagtat ccaacaagtg
     1981 ttgaaggaca ggtagttgaa gtaacgtatc ttccccctcg actggatcca tcaacaaggc
     2041 gaacaaatcc attcaacctc tcataaatta tctgatttac caaaccgata ccaaaatgac
     2101 cactcttgac gacacggctt accggtaccg caccagtgtc ccgggggacg ccgaggccat
     2161 cgaggcactg gatgggtcct tcaccaccga caccgtcttc cgcgtcaccg ccaccgggga
     2221 cggcttcacc ctgcgggagg tgccggtgga cccgcccctg accaaggtgt tccccgacga
     2281 cgaatcggac gacgaatcgg acgccgggga ggacggcgac ccggactccc ggacgttcgt
     2341 cgcgtacggg gacgacggcg acctggcggg cttcgtggtc gtctcgtact ccggctggaa
     2401 ccgccggctg accgtcgagg acatcgaggt cgccccggag caccgggggc acggggtcgg
     2461 gcgcgcgttg atggggctcg cgacggagtt cgcccgcgag cggggcgccg ggcacctctg
     2521 gctggaggtc accaacgtca acgcaccggc gatccacgcg taccggcgga tggggttcac
     2581 cctctgcggc ctggacaccg ccctgtacga cggcaccgcc tcggacggcg agcaggcgct
     2641 ctacatgagc atgccctgcc cctgagcgcg gccgcatact ggattggtga atcaatgagc
     2701 cgtagcacaa tggttacatt cggctagcta agatccaatg gcaaggacca agtgctggaa
     2761 cttgttttgc tttagcagat cttagcgtga gaggtatttg tcctctgtca ggagtagata
     2821 gtagatgttc tttttaaact aaaatgctaa ctgttccgaa ttcctcatcg cagctaatcc
     2881 gtacatcaaa agacaaaatg ctaggtatgt gtactacatc tcctgttgct agataagaca
     2941 tatgatagga aacacaccat caatagtcat tgtagcttta cttatactac gcatttgcac
     3001 tttcccctga gtggcagagg cgcattgaga aaatcgatct caacatagtt tatgtagcat
     3061 cccctagatc cattacttta agtctccttc gtctttggtg taggcatgtt ggacacaacg
     3121 aggtaaaaca caacacaaac aatgtgtcca gcaaagtagt agctgctcca gttctcccgc
     3181 gagcatcacg tgctataaaa ataattataa tttaaatttt ttaatataaa tatataaatt
     3241 aaaaatagaa agtaaaaaaa gaaattaaag aaaaaatagt ttttgttttc cgaagatgta
     3301 aaagactcta gggggatcgc caacaaatac taccttttat cttgctcttc ctgctctcag
     3361 gtattaatgc cgaattgttt catcttgtct gtgtagaaga ccacacacga aaatcctgtg
     3421 attttacatt ttacttatcg ttaatcgaat gtatatctat ttaatctgct tttcttgtct
     3481 aataaatata tatgtaaagt acgctttttg ttgaaatttt ttaaaccttt gtttattttt
     3541 ttttcttcat tccgtaactc ttctaccttc tttatttact ttctaaaatc caaatacaaa
     3601 acataaaaat aaataaacac agagtaaatt cccaaattat tccatcatta aaagatacga
     3661 ggcgcgtgta agttacaggc aagcgatcct agtacactct atattttttt atgcctcggt
     3721 aatgattttc attttttttt ttccacctag cggatgactc tttttttttc ttagcgattg
     3781 gcattatcac ataatgaatt atacattata taaagtaatg tgatttcttc gaagaatata
     3841 ctaaaaaatg agcaggcaag ataaacgaag gcaaagatga cagagcagaa agccctagta
     3901 aagcgtatta caaatgaaac caagattcag attgcgatct ctttaaaggg tggtccccta
     3961 gcgatagagc actcgatctt cccagaaaaa gaggcagaag cagtagcaga acaggccaca
     4021 caatcgcaag tgattaacgt ccacacaggt atagggtttc tggaccatat gatacatgct
     4081 ctggccaagc attccggctg gtcgctaatc gttgagtgca ttggtgactt acacatagac
     4141 gaccatcaca ccactgaaga ctgcgggatt gctctcggtc aagcttttaa agaggcccta
     4201 ctggcgcgtg gagtaaaaag gtttggatca ggatttgcgc ctttggatga ggcactttcc
     4261 agagcggtgg tagatctttc gaacaggccg tacgcagttg tcgaacttgg tttgcaaagg
     4321 gagaaagtag gagatctctc ttgcgagatg atcccgcatt ttcttgaaag ctttgcagag
     4381 gctagcagaa ttaccctcca cgttgattgt ctgcgaggca agaatgatca tcaccgtagt
     4441 gagagtgcgt tcaaggctct tgcggttgcc ataagagaag ccacctcgcc caatggtacc
     4501 aacgatgttc cctccaccaa aggtgttctt atgtagtttt acacaggagt ctggacttga
     4561 cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct
     4621 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4681 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4741 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4801 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4861 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4921 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4981 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5041 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     5101 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5161 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5221 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5281 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5341 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5401 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5461 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5521 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5581 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5641 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5701 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5761 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5821 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5881 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5941 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     6001 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     6061 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6121 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6181 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6241 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6301 ttttctgtga ctggtgagtc gatttattca acaaagccac gttgtgtctc aaaatctctg
     6361 atgttacatt gcacaagata aaaatatatc atcatgaaca ataaaactgt ctgcttacat
     6421 aaacagtaat acaaggggtg ttatgagcca tattcaacgg gaaacgtctt gctcgaggcc
     6481 gcgattaaat tccaacatgg atgctgattt atatgggtat aaatgggctc gcgataatgt
     6541 cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag cccgatgcgc cagagttgtt
     6601 tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca gatgagatgg tcagactaaa
     6661 ctggctgacg gaatttatgc ctcttccgac catcaagcat tttatccgta ctcctgatga
     6721 tgcatggtta ctcaccactg cgatccccgg gaaaacagca ttccaggtat tagaagaata
     6781 tcctgattca ggtgaaaata ttgttgatgc gctggcagtg ttcctgcgcc ggttgcattc
     6841 gattcctgtt tgtaattgtc cttttaacag cgatcgcgta tttcgtctcg ctcaggcgca
     6901 atcacgaatg aataacggtt tggttgatgc gagtgatttt gatgacgagc gtaatggctg
     6961 gcctgttgaa caagtctgga aagaaatgca taagcttttg ccattctcac cggattcagt
     7021 cgtcactcat ggtgatttct cacttgataa ccttattttt gacgagggga aattaatagg
     7081 ttgtattgat gttggacgag tcggaatcgc agaccgatac caggatcttg ccatcctatg
     7141 gaactgcctc ggtgagtttt ctccttcatt acagaaacgg ctttttcaaa aatatggtat
     7201 tgataatcct gatatgaata aattgcagtt tcatttgatg ctcgatgagt ttttctaatc
     7261 agaattggtt aattggttgt aacactggca ctcaaccaag tcattctgag aatagtgtat
     7321 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     7381 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     7441 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     7501 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     7561 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     7621 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     7681 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     7741 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
