LOCUS       40924_26196       10360 bp DNA     circular SYN 18-DEC-2018
DEFINITION  Gene deletion vector pJL2, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 10360)
  AUTHORS   Olson DG, Tripathi SA, Giannone RJ, Lo J, Caiazza NC, Hogsett DA, 
            Hettich RL, Guss AM, Dubrovsky G, Lynd LR.
  TITLE     Deletion of the Cel48S cellulase from Clostridium thermocellum
  JOURNAL   Proc. Natl. Acad. Sci. U.S.A. 107 (41), 17727-17732 (2010)
  PUBMED    20837514
REFERENCE   2  (bases 1 to 10360)
  AUTHORS   Olson DG.
  TITLE     Direct Submission
  JOURNAL   Submitted (18-AUG-2010) Thayer School of Engineering, Dartmouth 
            College, 8000 Cummings, Hanover, NH 03755, USA
REFERENCE   3  (bases 1 to 10360)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 10360)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Proc. Natl.
            Acad. Sci. U.S.A."; date: "2010"; volume: "107"; issue: "41"; pages:
            "17727-17732"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (18-AUG-2010) Thayer School of Engineering, Dartmouth College, 8000 
            Cummings, Hanover, NH 03755, USA"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..10360
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      27..274
                     /label=CYC1 terminator
                     /note="transcription terminator for CYC1"
     rep_origin      complement(522..1110)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     regulatory      1182..1526
                     /note="pfk; derived from Clostridium thermocellum strain
                     DSM 1313"
                     /regulatory_class="promoter"
     RBS             1512..1520
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             1527..2471
                     /codon_start=1
                     /product="pyrF"
                     /label=pyrF
                     /note="5FOA negative-selection in Clostridium thermocellum"
                     /protein_id="ADW95859.1"
                     /translation="MFIDTLIEKIREKDNPSVVGLDPKIEYVPSFIKEDMYKKYGKNLK
                     AVAEAILLFNKYIIDAVYDIVPAVKPQLAYYEMYGLEGMRVFYETCKYAKEKGLLVIAD
                     GKRNDIGSTAQCYSAAYLGKTDIDEGISEAVFDVDALTVNPYLGVDGIKPFIDDCVKYN
                     KGIFVLVKTSNKSSGEIQDILTQEGRSIYEIVAEYVESWGENKKGKYGYSCVGAVVGAT
                     YPNLAKILRKILKNSYILVPGYGAQGGTARDVAHCFNYDGLGAIVNASRSIMCAYKSEQ
                     WKNVYSEEKFYEASRAEAIRMRDDINSALRDRK"
     CDS             complement(2574..3431)
                     /label=AmpR
                     /note="beta-lactamase"
     CDS             complement(3530..4330)
                     /label=URA3
                     /note="orotidine-5'-phosphate decarboxylase, required for
                     uracil biosynthesis"
     promoter        complement(4331..4551)
                     /label=URA3 promoter
     misc_feature    4579..5082
                     /label=CEN/ARS
                     /note="S. cerevisiae CEN6 centromere fused to an
                     autonomously replicating sequence"
     CDS             complement(5314..6315)
                     /label=repB
                     /note="RepB replication protein"
     misc_feature    complement(6784..7783)
                     /note="cel48Y 3' flanking region; derived from Clostridium 
                     thermocellum strain DSM 1313"
     regulatory      7784..8431
                     /note="gapDH; derived from Clostridium thermocellum strain
                     DSM 1313"
                     /regulatory_class="promoter"
     CDS             8432..9223
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     misc_feature    complement(9227..10281)
                     /note="cel48Y 5' flanking region; derived from Clostridium 
                     thermocellum strain DSM 1313"
ORIGIN
        1 aaacccgctg atcctagagg gccgcatcat gtaattagtt atgtcacgct tacattcacg
       61 ccctcccccc acatccgctc taaccgaaaa ggaaggagtt agacaacctg aagtctaggt
      121 ccctatttat ttttttatag ttatgttagt attaagaacg ttatttatat ttcaaatttt
      181 tctttttttt ctgtacagac gcgtgtacgc atgtaacatt atactgaaaa ccttgcttga
      241 gaaggttttg ggacgctcga aggctttaat ttgcaagctg cggccctgca ttaatgaatc
      301 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
      361 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
      421 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
      481 caaaagccca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
      541 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
      601 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
      661 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
      721 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
      781 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
      841 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
      901 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
      961 aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     1021 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     1081 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     1141 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg ttgagccctt catccaagat
     1201 attttaaagc tcatttgagg gtagtcaagg gtaaagactt tgtcttatct atcgataacc
     1261 cttgactacc ctctgcactc gctcaagata gtagttaaga agggctaaaa gatgatgttg
     1321 tactttaaca cctaaaatta tggatgattt ccactatagg tgttgcattt aatattgcaa
     1381 tttacaaata aaaattaatt catatttttg gtttatgcag aattgcattt caaaatcaat
     1441 tatgttatat ttttatttag ttgtgtttaa aatttaacat tatagcagaa acacatatat
     1501 tttacatact ttaaggaggt tttaatatgt ttattgatac attaattgaa aagattagag
     1561 aaaaggataa tccttccgtt gtaggattag accctaaaat tgaatatgtt ccgtctttta
     1621 taaaggaaga catgtataaa aaatacggga aaaatttaaa agctgtggca gaggcgattc
     1681 tcctcttcaa taaatatatt attgatgcgg tttacgatat tgttcctgca gtaaaaccgc
     1741 agcttgcata ttatgaaatg tacggccttg aaggcatgag ggtgttttat gaaacttgca
     1801 aatatgcaaa ggaaaaagga cttttggtta ttgcagacgg aaaaagaaac gacataggtt
     1861 ccaccgccca gtgttattct gccgcatatc ttggaaaaac ggacattgat gaaggtataa
     1921 gcgaggcggt ttttgatgtg gatgccctga cagtcaaccc gtatcttggt gtggacggta
     1981 ttaagccttt tatagatgac tgtgtcaagt acaacaaggg catatttgtt ctggtcaaga
     2041 catcaaacaa gtcatccgga gaaattcagg acatactcac ccaggaagga agaagcattt
     2101 atgagattgt tgcggagtat gttgaatcat ggggtgaaaa caaaaaagga aaatatggat
     2161 acagttgtgt gggagcagtg gttggagcaa cttatcccaa tctggccaaa attttaagaa
     2221 agattctgaa aaattcctat atactggttc cgggctatgg agctcaggga ggaacagcca
     2281 gagatgtagc ccattgcttt aattatgacg ggctcggagc aattgtcaat gcatcaagaa
     2341 gcataatgtg tgcctacaaa tctgaacaat ggaagaatgt ttacagcgaa gaaaagtttt
     2401 atgaggcatc aagagccgag gcaataagaa tgagggacga tattaacagt gcgttgcgag
     2461 acaggaagta acatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     2521 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     2581 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     2641 ctccccgtcg tgtagataac tacgatacgg gagcgcttac catctggccc cagtgctgca
     2701 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     2761 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     2821 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttggc
     2881 attgctacag gcatcgtggt gtcactctcg tcgtttggta tggcttcatt cagctccggt
     2941 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     3001 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     3061 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     3121 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     3181 gcgtcaatac gggataatag tgtatcacat agcagaactt taaaagtgct catcattgga
     3241 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     3301 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     3361 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     3421 tgaatactca tactcttcct ttttcaatgg gtaataactg atataattaa attgaagctc
     3481 taatttgtga gtttagtata catgcattta cttataatac agttttttag ttttgctggc
     3541 cgcatcttct caaatatgct tcccagcctg cttttctgta acgttcaccc tctaccttag
     3601 catcccttcc ctttgcaaat agtcctcttc caacaataat aatgtcagat cctgtagaga
     3661 ccacatcatc cacggttcta tactgttgac ccaatgcgtc tcccttgtca tctaaaccca
     3721 caccgggtgt cataatcaac caatcgtaac cttcatctct tccacccatg tctctttgag
     3781 caataaagcc gataacaaaa tctttgtcgc tcttcgcaat gtcaacagta cccttagtat
     3841 attctccagt agatagggag cccttgcatg acaattctgc taacatcaaa aggcctctag
     3901 gttcctttgt tacttcttct gccgcctgct tcaaaccgct aacaatacct gggcccacca
     3961 caccgtgtgc attcgtaatg tctgcccatt ctgctattct gtatacaccc gcagagtact
     4021 gcaatttgac tgtattacca atgtcagcaa attttctgtc ttcgaagagt aaaaaattgt
     4081 acttggcgga taatgccttt agcggcttaa ctgtgccctc catggaaaaa tcagtcaaga
     4141 tatccacatg tgtttttagt aaacaaattt tgggacctaa tgcttcaact aactccagta
     4201 attccttggt ggtacgaaca tccaatgaag cacacaagtt tgtttgcttt tcgtgcatga
     4261 tattaaatag cttggcagca acaggactag gatgagtagc agcacgttcc ttatatgtag
     4321 ctttcgacat gatttatctt cgtttcctgc aggtttttgt tctgtgcagt tgggttaaga
     4381 atactgggca atttcatgtt tcttcaacac tacatatgcg tatatatacc aatctaagtc
     4441 tgtgctcctt ccttcgttct tccttctgtt cggagattac cgaatcaaaa aaatttcaaa
     4501 gaaaccgaaa tcaaaaaaaa gaataaaaaa aaaatgatga attgaattga aaagctagct
     4561 tatcgatggg tccttttcat cacgtgctat aaaaataatt ataatttaaa ttttttaata
     4621 taaatatata aattaaaaat agaaagtaaa aaaagaaatt aaagaaaaaa tagtttttgt
     4681 tttccgaaga tgtaaaagac tctaggggga tcgccaacaa atactacctt ttatcttgct
     4741 cttcctgctc tcaggtatta atgccgaatt gtttcatctt gtctgtgtag aagaccacac
     4801 acgaaaatcc tgtgatttta cattttactt atcgttaatc gaatgtatat ctatttaatc
     4861 tgcttttctt gtctaataaa tatatatgta aagtacgctt tttgttgaaa ttttttaaac
     4921 ctttgtttat ttttttttct tcattccgta actcttctac cttctttatt tactttctaa
     4981 aatccaaata caaaacataa aaataaataa acacagagta aattcccaaa ttattccatc
     5041 attaaaagat acgaggcgcg tgtaagttac aggcaagcga tccgtccgcc ggcgaacgtg
     5101 gcgagaaagg aagggaagaa agcgaaagga gcgggggcta gggcggtggg aagtgtaggg
     5161 gtcacgctgg gcgtaaccac cacacccgcc gcgcttaatg gggcgctaca gggcgcgtgg
     5221 ggatgatcca ctagtgaatt taggaggctt acttgtctgc tttcttcatt agaatcaatc
     5281 cttttttaaa agtcaatccc gtttgttgaa ctactcttta ataaaataat ttttccgttc
     5341 ccaattccac attgcaataa tagaaaatcc atcttcatcg gctttttcgt catcatctgt
     5401 atgaatcaaa tcgccttctt ctgtgtcatc aaggtttaat tttttatgta tttcttttaa
     5461 caaaccacca taggagatta accttttacg gtgtaaacct tcctccaaat cagacaaacg
     5521 tttcaaattc ttttcttcat catcggtcat aaaatccgta tcctttacag gatattttgc
     5581 agtttcgtca attgccgatt gtatatccga tttatattta tttttcggtc gaatcatttg
     5641 aacttttaca tttggatcat agtctaattt cattgccttt ttccaaaatt gaatccattg
     5701 tttttgattc acgtagtttt ctgtattctt aaaataagtt ggttccacac ataccaatac
     5761 atgcatgtgc tgattataag aattatcttt attatttatt gtcacttccg ttgcacgcat
     5821 aaaaccaaca agatttttat taattttttt atattgcatc attcggcgaa atccttgagc
     5881 catatctgac aaactcttat ttaattcttc gccatcataa acatttttaa ctgttaatgt
     5941 gagaaacaac caacgaactg ttggcttttg tttaataact tcagcaacaa ccttttgtga
     6001 ctgaatgcca tgtttcattg ctctcctcca gttgcacatt ggacaaagcc tggatttaca
     6061 aaaccacact cgatacaact ttctttcgcc tgtttcacga ttttgtttat actctaatat
     6121 ttcagcacaa tcttttactc tttcagcctt tttaaattca agaatatgca gaagttcaaa
     6181 gtaatcaaca ttagcgattt tcttttctct ccatggtctc acttttccac tttttgtctt
     6241 gtccactaaa acccttgatt tttcatctga ataaatgcta ctattaggac acataatatt
     6301 aaaagaaacc cccatctatt tagttatttg tttggtcact tataacttta acagatgggg
     6361 tttttctgtg caaccaattt taagggtttt caatacttta aaacacatac ataccaacac
     6421 ttcaacgcac ctttcagcaa ctaaaataaa aatgacgtta tttctatatg tatcaagaat
     6481 agaaagaact cgtttttcgc tacgctcaaa acgcaaaaaa agcactcatt cgagtgcttt
     6541 ttcttatcgc tccaaatcat gcgatttttt cctctttgct tttctttgct cacgaagttc
     6601 tcgatcacgc tgcaaaacat cttgaagcga aaaagtattc ttcttttctt ccgatcgctc
     6661 atgctgacgc acgaaaagcc ctctaggcgc ataggaacaa ctcctaaatg catgtgaggg
     6721 gttttctcgt ccatgtgaac agtcgcatac gcaatatttt gtttcccata gctagtaagc
     6781 ttgtatcaca ggaagtgacg cggaaggggc aggacagatg ttcaaggtta caacccttga
     6841 tataaacaac cctcccagaa aagaagacgg aacggtggat ttttcaaaag acttttttga
     6901 cagggaaaca aatcttactg taagcggaca gcttgagggt gaaacataca gcatggcctt
     6961 tagaaatata tacactttcg ggccgacttt cagagcggag aactccaata cggcaaggca
     7021 tgcggcggaa ttttggatgg tggagccgga aatagccttt gccgatcttg aggatgacat
     7081 ggaacttgca gaggacatgc tcaagtatat cataaattac tgcctggaaa atgctccgga
     7141 ggaaatggag ttctttaaca attttattga caatacactg cttgatcggc tcaacaatat
     7201 tgtaagttct gattttgccc atgttacata taccgaggcc attgatattt tgtcaaaagc
     7261 cgatcaaaaa tttgagtatc ctgtaaagtg gggtaacgac ctgcagaccg aacatgaaag
     7321 ataccttacg gaaaaggtgt ttaaaaagcc tgtgtttgtg actgactatc caaaagatat
     7381 aaaagccttt tacatgaggc tgaatgacga taacaaaacc gttgcggcaa tggaccttct
     7441 tgtgccaggt gtcggagaga taatcggagg aagccagagg gaagagagac tggattattt
     7501 ggaaaacaga atgaaagagc ttggacttaa aatggaggat tactggtggt acttggatct
     7561 tcgcaggtat ggaagcacca gacatgcagg cttcggactg ggatttgaaa gggctataat
     7621 gtatatcacc ggcatgtcaa acataagaga tgtaatacct ttcccgagaa ctgtgcactc
     7681 agcggaattt taacggaaat tccgttcaaa aagatgtaaa aaagaaaggg gcggtttttg
     7741 tgatgcactt ttataaaaac cgtcccttgt gttttattgc atcccagtat tctgacatgg
     7801 gtgtatcaat aacccatgcg tttccgtatt gtatcggaat ggtttcggac agggcggtgg
     7861 gaatagacat ggaaaagatt tttttgcccg aggatgcatt gataaagtat ttcttttccg
     7921 aaagagagga aaagattcta aagagttttg gaaatactga tgaatattgt gtgcagagta
     7981 caattctatg gacaagaaaa gaggctttgt caaaactttt tcgtctggga atgaggatgg
     8041 attttaaaaa gctggatact ttggaggacg aggtggtttt tcaggaaaca aacagggcgc
     8101 gtctgttttc ttttatatgc aataattact gtatctctct ggcattgcca ggttttaata
     8161 aagattaaaa ttattgacta gaaataaaaa aattgtccat aatattaatg gacaaaaaaa
     8221 caaagaatta catcaaagga agataaaaat actttgttaa aaaattaatt attttttatc
     8281 taaactattg aaaatgaaaa taaaataata taaaatgaat catagtgcaa gagatacttg
     8341 ccagaggatg aatattttac tgcattcatg ctttatggca gctaatagag gcattaaatt
     8401 aaattttaat ttacaatagg aggcgatatt aatggctaaa atgagaatat caccggaatt
     8461 gaaaaaactg atcgaaaaat accgctgcgt aaaagatacg gaaggaatgt ctcctgctaa
     8521 ggtatataag ctggtgggag aaaatgaaaa cctatattta aaaatgacgg acagccggta
     8581 taaagggacc acctatgatg tggaacggga aaaggacatg atgctatggc tggaaggaaa
     8641 gctgcctgtt ccaaaggtcc tgcactttga acggcatgat ggctggagca atctgctcat
     8701 gagtgaggcc gatggcgtcc tttgctcgga agagtatgaa gatgaacaaa gccctgaaaa
     8761 gattatcgag ctgtatgcgg agtgcatcag gctctttcac tccatcgaca tatcggattg
     8821 tccctatacg aatagcttag acagccgctt agccgaattg gattacttac tgaataacga
     8881 tctggccgat gtggattgcg aaaactggga agaagacact ccatttaaag atccgcgcga
     8941 gctgtatgat tttttaaaga cggaaaagcc cgaagaggaa cttgtctttt cccacggcga
     9001 cctgggagac agcaacatct ttgtgaaaga tggcaaagta agtggcttta ttgatcttgg
     9061 gagaagcggc agggcggaca agtggtatga cattgccttc tgcgtccggt cgatcaggga
     9121 ggatatcggg gaagaacagt atgtcgagct attttttgac ttactgggga tcaagcctga
     9181 ttgggagaaa ataaaatatt atattttact ggatgaattg ttttaggatc ctcccctgat
     9241 aaacctcctt ttataaactt ttataaatat ttacctcatg taattattat actaggtaac
     9301 ttaaaccatt acaaatattt ctcttgcaga ggcggtaaga attattttta tttttaaaat
     9361 taatttttaa aacttttttt tgtgttgtcc gtatatatga tatgaaacaa aagtgaattg
     9421 atagtactgt cataaaggcg tttatgaagc atagaggagg agaggttgat gaaaaacggt
     9481 ttggtccgtt caaagtatga cagggttata tcaggtgtgt gtgggggaat aggagaatat
     9541 tttaatattg accctaccat tgtaaggctt ggttttgtta tagcgactgt tttatttagt
     9601 ggccttgggc tgtttgcgta tattgtggca atttttgtaa tacccgaagc tgacagcgca
     9661 gccaatggct ttactgatta tgcccaaaaa ggatttaacg attattatga ccctgacaag
     9721 gatttcagca aagtattgga agatgatgag gaatatataa aaaaggattc tcatggcgga
     9781 aaaacctttg ttggcatttg tctcattgtc cttggacttg tctccctgtt aagagaacat
     9841 tttagggaat atattagcaa atttattcac attgatttta aaattttagt gtctttgctg
     9901 ctgatagttg ttggagcgtt gattatattc agaggcagga ggcgtttgcc atgaaaaaga
     9961 ataacagtgg catatttttt ggacttttta taatttttat tggtatagtg ctcatactat
    10021 ctcagtttgg tatgctggac cttgacaaaa tagtgaattt cctttcagat catatatcaa
    10081 ctataatctc tttgctgctt attgtgacgg gagtaaacct tgtttttgac aagtataatg
    10141 gggttaaatt ctttgcgtgg acggctttct ttgcagtact gttaatatcc ggctgcatct
    10201 atggtggtgc aacggaaaat aagattgaga gaagcactaa taaggcctat atcagaccct
    10261 ttactgagga aaagctgccc ggggcctcga gaaaacaaaa ggctcagtcg gaagactggg
    10321 ccttttgttt tggtaccgaa ttcggcgcgc ctcagcgttt
//
