LOCUS       sequence-181275-        8601 bp    DNA     circular SYN 14-APR-2025
DEFINITION  For gene disruption in Clostridium acetobutylicum ATCC 824 and 
            Clostridium beijerinckii NCIMB8052.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8601)
  AUTHORS   Zhang N, Shao L, Jiang Y, Gu Y, Li Q, Liu J, Jiang W, Yang S
  TITLE     I-SceI-mediated scarless gene modification via allelic exchange in 
            Clostridium.
  JOURNAL   J Microbiol Methods. 2015 Jan;108:49-60. doi: 
            10.1016/j.mimet.2014.11.004. Epub 2014 Nov 15.
  PUBMED    25451462
REFERENCE   2  (bases 1 to 8601)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Microbiol
            Methods."; date: "2015-01"; volume: "108:49-60. doi"; pages: " 
            10.1016/j.mimet.2014.11.004. Epub 2014 Nov 15"
COMMENT     Sequence Label: pWJ1
FEATURES             Location/Qualifiers
     source          1..8601
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      116..202
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      294..321
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     complement(379..398)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     498..520
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(558..576)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        644..748
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(749..817,818..1609)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(967..986)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1780..2368
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2269..2288
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2522..2539
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2656..2677
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2692..2697,2698..2715,2716..2722)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2730..2746
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
     primer_bind     2735..2757
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2754..2770
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2754..2770
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     RBS             6519..6541
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 acttggagcc actatcgact acgcgatcat ggcgaccaca cccgtcctgt ggatcgccaa
       61 gccgccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca ggcatcaaat
      121 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
      181 cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga agcaacggcc
      241 cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta agcagaaggc
      301 catcctgacg gatggccttt ttgcgtttct acaaactctt cctgtcgtca tatctacaag
      361 catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
      421 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
      481 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
      541 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
      601 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      661 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
      721 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
      781 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
      841 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
      901 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
      961 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     1021 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     1081 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     1141 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     1201 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     1261 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     1321 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1381 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1441 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     1501 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1561 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1621 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1681 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     1741 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     1801 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     1861 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     1921 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     1981 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2041 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2101 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2161 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2221 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2281 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2341 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2401 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2461 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2521 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     2581 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     2641 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     2701 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     2761 cagctatgac catgattacg ccaagctttg gctaacacac acgccattcc aaccaatagt
     2821 tttctcggca taaagccatg ctctgacgct taaatgcact aatgccttaa aaaaacatta
     2881 aagtctaaca cactagactt atttacttcg taattaagtc gttaaaccgt gtgctctacg
     2941 accaaaagta taaaaccttt aagaactttc ttttttcttg taaaaaaaga aactagataa
     3001 atctctcata tcttttattc aataatcgca tcagattgca gtataaattt aacgatcact
     3061 catcatgttc atatttatca gagctcgtgc tataattata ctaattttat aaggaggaaa
     3121 aaataaagag ggttataatg aacgagaaaa atataaaaca cagtcaaaac tttattactt
     3181 caaaacataa tatagataaa ataatgacaa atataagatt aaatgaacat gataatatct
     3241 ttgaaatcgg ctcaggaaaa gggcatttta cccttgaatt agtacagagg tgtaatttcg
     3301 taactgccat tgaaatagac cataaattat gcaaaactac agaaaataaa cttgttgatc
     3361 acgataattt ccaagtttta aacaaggata tattgcagtt taaatttcct aaaaaccaat
     3421 cctataaaat atttggtaat ataccttata acataagtac ggatataata cgcaaaattg
     3481 tttttgatag tatagctgat gagatttatt taatcgtgga atacgggttt gctaaaagat
     3541 tattaaatac aaaacgctca ttggcattat ttttaatggc agaagttgat atttctatat
     3601 taagtatggt tccaagagaa tattttcatc ctaaacctaa agtgaatagc tcacttatca
     3661 gattaaatag aaaaaaatca agaatatcac acaaagataa acagaagtat aattatttcg
     3721 ttatgaaatg ggttaacaaa gaatacaaga aaatatttac aaaaaatcaa tttaacaatt
     3781 ccttaaaaca tgcaggaatt gacgatttaa acaatattag ctttgaacaa ttcttatctc
     3841 ttttcaatag ctataaatta tttaataagt aagttaaggg atgcataaac tgcatccctt
     3901 aacttgtttt tcgtgtacct attttttgtg aatcgataat ttacagaaaa gaaaattata
     3961 gaatttagta tgattaatta tactcattta tgaatgttta attgaataca aaaaaaaata
     4021 cttgttatgt attcaattac gggttaaaat atagacaagt tgaaaaattt aataaaaaaa
     4081 taagtcctca gctcttatat attaagctac caacttagta tataagccaa aacttaaatg
     4141 tgctaccaac acatcaagcc gttagagaac tctatctata gcaatatttc aaatgtaccg
     4201 acatacaaga gaaacattaa ctatatatat tcaatttatg agattatctt aacagatata
     4261 aatgtaaatt gcaataagta agatttagaa gtttatagcc tttgtgtatt ggaagcagta
     4321 cgcaaaggct tttttatttg ataaaaatta gaagtatatt tattttttca taattaattt
     4381 atgaaaatga aagggggtga gcaaagtgac agaggaaagc agtatcttat caaataacaa
     4441 ggtattagca atatcattat tgactttagc agtaaacatt atgactttta tagtgcttgt
     4501 agctaagtag tacgaaaggg ggagctttaa aaagctcctt ggaatacata gaattcataa
     4561 attaatttat gaaaagaagg gcgtatatga aaacttgtaa aaattgcaaa gagtttatta
     4621 aagatactga aatatgcaaa atacattcgt tgatgattca tgataaaaca gtagcaacct
     4681 attgcagtaa atacaatgag tcaagatgtt tacataaagg gaaagtccaa tgtattaatt
     4741 gttcaaagat gaaccgatat ggatggtgtg ccataaaaat gagatgtttt acagaggaag
     4801 aacagaaaaa agaacgtaca tgcattaaat attatgcaag gagctttaaa aaagctcatg
     4861 taaagaagag taaaaagaaa aaataattta tttattaatt taatattgag agtgccgaca
     4921 cagtatgcac taaaaaatat atctgtggtg tagtgagccg atacaaaagg atagtcactc
     4981 gcattttcat aatacatctt atgttatgat tatgtgtcgg tgggacttca cgacgaaaac
     5041 ccacaataaa aaaagagttc ggggtagggt taagcatagt tgaggcaact aaacaatcaa
     5101 gctaggatat gcagtagcag accgtaaggt cgttgtttag gtgtgttgta atacatacgc
     5161 tattaagatg taaaaatacg gataccaatg aagggaaaag tataattttt ggatgtagtt
     5221 tgtttgttca tctatgggca aactacgtcc aaagccgttt ccaaatctgc taaaaagtat
     5281 atcctttcta aaatcaaagt caagtatgaa atcataaata aagtttaatt ttgaagttat
     5341 tatgatatta tgtttttcta ttaaaataaa ttaagtatat agaatagttt aataatagta
     5401 tatacttaat gtgctgcagt ataaaatata aataattttc taaaaaactt aacttcatgt
     5461 gaaaagtttg ttaaaatata aatgagcacg ttaatcattt aacatagata attaaatagt
     5521 aaaagggagt gtcgaggatc ctcgagataa ttatccttaa aggacgtaga tgtgcgccca
     5581 gatagggtgt taagtcaagt agtttaaggt actactctgt aagataacac agaaaacagc
     5641 caacctaacc gaaaagcgaa agctgatacg ggaacagagc acggttggaa agcgatgagt
     5701 tacctaaaga caatcgggta cgactgagtc gcaatgttaa tcagatataa ggtataagtt
     5761 gtgtttactg aacgcaagtt tctaatttcg gtttccttcc gatagaggaa agtgtctgaa
     5821 acctctagta caaagaaagg taagttaaca tctacgactt atctgttatc accacatttg
     5881 tacaatctgt aggagaacct atgggaacga aacgaaagcg atgccgagaa tctgaattta
     5941 ccaagactta acactaactg gggataccct aaacaagaat gcctaataga aaggaggaaa
     6001 aaggctatag cactagagct tgaaaatctt gcaagggtac ggagtactcg tagtagtctg
     6061 agaagggtaa cgccctttac atggcaaagg ggtacagtta ttgtgtacta aaattaaaaa
     6121 ttgattaggg aggaaaacct caaaatgaaa ccaacaatgg caattttaga aagaatcagt
     6181 aaaaattcac aagaaaatat agacgaagtt tttacaagac tttatcgtta tcttttacgt
     6241 ccagatattt attacgtggc gacgcgttgg gaaatggcaa tgatagcgaa acaacgtaaa
     6301 actcttgttg tatgctttca ttgtcatcgt cacgtgattc ataaacacaa gtgaatgtcg
     6361 acagtgaatt tttacgaacg aacaataaca gagccgtata ctccgagagg ggtacgtacg
     6421 gttcccgaag agggtggtgc aaaccagtca cagtaatgtg aacaaggcgg tacctcccta
     6481 cttcaccata tcattttctg cagcccccta gaaataattt tgtttaactt taagaaggag
     6541 atatacatat atggctagat cgtccattcc gacagcatcg ccagtcacta tggcgtgctg
     6601 ctagcgctat atgcgttgat gcaatttcta tgcactcgta gtagtctgag aagggtaacg
     6661 ccctttacat ggcaaagggg tacagttatt gtgtactaaa attaaaaatt gattagggag
     6721 gaaaacctca aaatgaaacc aacaatggca attttagaaa gaatcagtaa aaattcacaa
     6781 gaaaatatag acgaagtttt tacaagactt tatcgttatc ttttacgtcc agatatttat
     6841 tacgtggcgt atcaaaattt atattccaat aaaggagctt ccacaaaagg aatattagat
     6901 gatacagcgg atggctttag tgaagaaaaa ataaaaaaga ttattcaatc tttaaaagac
     6961 ggaacttact atcctcaacc tgtacgaaga atgtatattg caaaaaagaa ttctaaaaag
     7021 atgagacctt taggaattcc aactttcaca gataaattga tccaagaagc tgtgagaata
     7081 attcttgaat ctatctatga accggtattc gaagatgtgt ctcacggttt tagacctcaa
     7141 cgaagctgtc acacagcttt gaaaacaatc aaaagagagt ttggcggcgc aagatggttt
     7201 gtggagggag atataaaagg ctgcttcgat aatatagacc acgttacact cattggactc
     7261 atcaatctta aaatcaaaga tatgaaaatg agccaattga tttataaatt tctaaaagca
     7321 ggttatctgg aaaactggca gtatcacaaa acttacagcg gaacacctca aggtggaatt
     7381 ctatctcctc ttttggccaa catctatctt catgaattgg ataagtttgt tttacaactc
     7441 aaaatgaagt ttgaccgaga aagtccagaa agaataacac ctgaatatcg ggagctccac
     7501 aatgagataa aaagaatttc tcaccgtctc aagaagttgg agggtgaaga aaaagctaaa
     7561 gttcttttag aatatcaaga aaaacgtaaa agattaccca cactcccctg tacctcacag
     7621 acaaataaag tattgaaata cgtccggtat gcggacgact tcattatctc tgttaaagga
     7681 agcaaagagg actgtcaatg gataaaagaa caattaaaac tttttattca taacaagcta
     7741 aaaatggaat tgagtgaaga aaaaacactc atcacacata gcagtcaacc cgctcgtttt
     7801 ctgggatatg atatacgagt aaggagatct ggaacgataa aacgatctgg taaagtcaaa
     7861 aagagaacac tcaatgggag tgtagaactc cttattcctc ttcaagacaa aattcgtcaa
     7921 tttatttttg acaagaaaat agctatccaa aagaaagata gctcatggtt tccagttcac
     7981 aggaaatatc ttattcgttc aacagactta gaaatcatca caatttataa ttctgaactc
     8041 cgcgggattt gtaattacta cggtctagca agtaatttta accagctcaa ttattttgct
     8101 tatcttatgg aatacagctg tctaaaaacg atagcctcca aacataaggg aacactttca
     8161 aaaaccattt ccatgtttaa agatggaagt ggttcgtggg ggatcccgta tgagataaag
     8221 caaggtaagc agcgccgtta ttttgcaaat tttagtgaat gtaaatcccc ttatcaattt
     8281 acggatgaga taagtcaagc tcctgtattg tatggctatg cccggaatac tcttgaaaac
     8341 aggttaaaag ctaaatgttg tgaattatgt gggacgtctg atgaaaatac ttcctatgaa
     8401 attcaccatg tcaataaggt caaaaatctt aaaggcaaag aaaaatggga aatggcaatg
     8461 atagcgaaac aacgtaaaac tcttgttgta tgctttcatt gtcatcgtca cgtgattcat
     8521 aaacacaagt gaatgtcgag cacccgttct cggagcactg tccgaccgct ttggccgccg
     8581 cccagtcctg ctcgcttcgc t
//
