LOCUS       40924_9396        8519 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Cloning vector pCDF-Crispr-CasWT DNA, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8519)
  AUTHORS   Hamamoto K, Tokunaga T, Yagi Y, Hirai I.
  TITLE     Characterization of blaCTX-M-14 transposition from plasmid to 
            chromosome in Escherichia coli model strain
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8519)
  AUTHORS   Hamamoto K, Hirai I.
  TITLE     Direct Submission
  JOURNAL   Submitted (22-MAR-2018) Contact:Kouta Hamamoto University of the 
            Ryukyus, Microbiology; 207 Uehara, Nishihara-cho, Nakagami-gun, 
            Okinawa 903-0215, Japan
REFERENCE   3  (bases 1 to 8519)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 8519)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (22-MAR-2018) Contact:Kouta Hamamoto University of the Ryukyus, 
            Microbiology; 207 Uehara, Nishihara-cho, Nakagami-gun, Okinawa 
            903-0215, Japan"
COMMENT     SGRef: number: 3; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..8519
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(61..684)
                     /label=TetR
                     /note="tetracycline repressor TetR"
     promoter        700..755
                     /label=tetR/tetA promoters
                     /note="overlapping promoters for bacterial tetR and tetA"
     RBS             773..784
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             791..4894
                     /label=Cas9
                     /note="Cas9 (Csn1) endonuclease from the Streptococcus
                     pyogenes Type II CRISPR/Cas system"
     terminator      4921..4992
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
     terminator      5015..5062
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     CDS             complement(5236..6024)
                     /label=SmR
                     /note="aminoglycoside adenylyltransferase (Murphy, 1985)"
     promoter        complement(6025..6116)
                     /label=AmpR promoter
     rep_origin      complement(6164..6902)
                     /direction=LEFT
                     /label=CloDF13 ori
                     /note="Plasmids containing the CloDF13 (CDF) origin of 
                     replication can be propagated in E. coli cells that contain
                     additional plasmids with compatible origins."
     misc_RNA        complement(6939..7014)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    complement(7044..7060)
                     /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(7068..7097)
                     /label=trc promoter
                     /note="strong E. coli promoter; hybrid between the trp and
                     lac UV5 promoters"
     CDS             complement(7332..8411)
                     /label=lacI
                     /note="lac repressor"
     promoter        complement(8412..8489)
                     /label=lacI promoter
ORIGIN
        1 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agcatgctta
       61 agacccactt tcacatttaa gttgtttttc taatccgcat atgatcaatt caaggccgaa
      121 taagaaggct ggctctgcac cttggtgatc aaataattcg atagcttgtc gtaataatgg
      181 cggcatacta tcagtagtag gtgtttccct ttcttcttta gcgacttgat gctcttgatc
      241 ttccaatacg caacctaaag taaaatgccc cacagcgctg agtgcatata atgcattctc
      301 tagtgaaaaa ccttgttggc ataaaaaggc taattgattt tcgagagttt catactgttt
      361 ttctgtaggc cgtgtaccta aatgtacttt tgctccatcg cgatgactta gtaaagcaca
      421 tctaaaactt ttagcgttat tacgtaaaaa atcttgccag ctttcccctt ctaaagggca
      481 aaagtgagta tggtgcctat ctaacatctc aatggctaag gcgtcgagca aagcccgctt
      541 attttttaca tgccaataca atgtaggctg ctctacacct agcttctggg cgagtttacg
      601 ggttgttaaa ccttcgattc cgacctcatt aagcagctct aatgcgctgt taatcacttt
      661 acttttatct aatctagaca tcattaattc ctaatttttg ttgacactct atcgttgata
      721 gagttatttt accactccct atcagtgata gagaaaagaa ttcaaaagat ctaaagagga
      781 gaaaggatct atggataaga aatactcaat aggcttagat atcggcacaa atagcgtcgg
      841 atgggcggtg atcactgatg aatataaggt tccgtctaaa aagttcaagg ttctgggaaa
      901 tacagaccgc cacagtatca aaaaaaatct tataggggct cttttatttg acagtggaga
      961 gacagcggaa gcgactcgtc tcaaacggac agctcgtaga aggtatacac gtcggaagaa
     1021 tcgtatttgt tatctacagg agattttttc aaatgagatg gcgaaagtag atgatagttt
     1081 ctttcatcga cttgaagagt cttttttggt ggaagaagac aagaagcatg aacgtcatcc
     1141 tatttttgga aatatagtag atgaagttgc ttatcatgag aaatatccaa ctatctatca
     1201 tctgcgaaaa aaattggtag attctactga taaagcggat ttgcgcttaa tctatttggc
     1261 cttagcgcat atgattaagt ttcgtggtca ttttttgatt gagggagatt taaatcctga
     1321 taatagtgat gtggacaaac tatttatcca gttggtacaa acctacaatc aattatttga
     1381 agaaaaccct attaacgcaa gtggagtaga tgctaaagcg attctttctg cacgattgag
     1441 taaatcaaga cgattagaaa atctcattgc tcagctcccc ggtgagaaga aaaatggctt
     1501 atttgggaat ctcattgctt tgtcattggg tttgacccct aattttaaat caaattttga
     1561 tttggcagaa gatgctaaat tacagctttc aaaagatact tacgatgatg atttagataa
     1621 tttattggcg caaattggag atcaatatgc tgatttgttt ttggcagcta agaatttatc
     1681 agatgctatt ttactttcag atatcctaag agtaaatact gaaataacta aggctcccct
     1741 atcagcttca atgattaaac gctacgatga acatcatcaa gacttgactc ttttaaaagc
     1801 tttagttcga caacaacttc cagaaaagta taaagaaatc ttttttgatc aatcaaaaaa
     1861 cggatatgca ggttatattg atgggggagc tagccaagaa gaattttata aatttatcaa
     1921 accaatttta gaaaaaatgg atggtactga ggaattattg gtgaaactaa atcgtgaaga
     1981 tttgctgcgc aagcaacgga cctttgacaa cggctctatt ccccatcaaa ttcacttggg
     2041 tgagctgcat gctattttga gaagacaaga agacttttat ccatttttaa aagacaatcg
     2101 tgagaagatt gaaaaaatct tgacttttcg aattccttat tatgttggtc cattggcgcg
     2161 tggcaatagt cgttttgcat ggatgactcg gaagtctgaa gaaacaatta ccccatggaa
     2221 ttttgaagaa gttgtcgata aaggtgcttc agctcaatca tttattgaac gcatgacaaa
     2281 ctttgataaa aatcttccaa atgaaaaagt actaccaaaa catagtttgc tttatgagta
     2341 ttttacggtt tataacgaat tgacaaaggt caaatatgtt actgaaggaa tgcgaaaacc
     2401 agcatttctt tcaggtgaac agaagaaagc cattgttgat ttactcttca aaacaaatcg
     2461 aaaagtaacc gttaagcaat taaaagaaga ttatttcaaa aaaatagaat gttttgatag
     2521 tgttgaaatt tcaggagttg aagatagatt taatgcttca ttaggtacct accatgattt
     2581 gctaaaaatt attaaagata aagatttttt ggataatgaa gaaaatgaag atatcttaga
     2641 ggatattgtt ttaacattga ccttatttga agatagggag atgattgagg aaagacttaa
     2701 aacatatgct cacctctttg atgataaggt gatgaaacag cttaaacgtc gccgttatac
     2761 tggttgggga cgtttgtctc gaaaattgat taatggtatt agggataagc aatctggcaa
     2821 aacaatatta gattttttga aatcagatgg ttttgccaat cgcaatttta tgcagctgat
     2881 ccatgatgat agtttgacat ttaaagaaga cattcaaaaa gcacaagtgt ctggacaagg
     2941 cgatagttta catgaacata ttgcaaattt agctggtagc cctgctatta aaaaaggtat
     3001 tttacagact gtaaaagttg ttgatgaatt ggtcaaagta atggggcggc ataagccaga
     3061 aaatatcgtt attgaaatgg cacgtgaaaa tcagacaact caaaagggcc agaaaaattc
     3121 gcgagagcgt atgaaacgaa tcgaagaagg tatcaaagaa ttaggaagtc agattcttaa
     3181 agagcatcct gttgaaaata ctcaattgca aaatgaaaag ctctatctct attatctcca
     3241 aaatggaaga gacatgtatg tggaccaaga attagatatt aatcgtttaa gtgattatga
     3301 tgtcgatcac attgttccac aaagtttcct taaagacgat tcaatagaca ataaggtctt
     3361 aacgcgttct gataaaaatc gtggtaaatc ggataacgtt ccaagtgaag aagtagtcaa
     3421 aaagatgaaa aactattgga gacaacttct aaacgccaag ttaatcactc aacgtaagtt
     3481 tgataattta acgaaagctg aacgtggagg tttgagtgaa cttgataaag ctggttttat
     3541 caaacgccaa ttggttgaaa ctcgccaaat cactaagcat gtggcacaaa ttttggatag
     3601 tcgcatgaat actaaatacg atgaaaatga taaacttatt cgagaggtta aagtgattac
     3661 cttaaaatct aaattagttt ctgacttccg aaaagatttc caattctata aagtacgtga
     3721 gattaacaat taccatcatg cccatgatgc gtatctaaat gccgtcgttg gaactgcttt
     3781 gattaagaaa tatccaaaac ttgaatcgga gtttgtctat ggtgattata aagtttatga
     3841 tgttcgtaaa atgattgcta agtctgagca agaaataggc aaagcaaccg caaaatattt
     3901 cttttactct aatatcatga acttcttcaa aacagaaatt acacttgcaa atggagagat
     3961 tcgcaaacgc cctctaatcg aaactaatgg ggaaactgga gaaattgtct gggataaagg
     4021 gcgagatttt gccacagtgc gcaaagtatt gtccatgccc caagtcaata ttgtcaagaa
     4081 aacagaagta cagacaggcg gattctccaa ggagtcaatt ttaccaaaaa gaaattcgga
     4141 caagcttatt gctcgtaaaa aagactggga tccaaaaaaa tatggtggtt ttgatagtcc
     4201 aacggtagct tattcagtcc tagtggttgc taaggtggaa aaagggaaat cgaagaagtt
     4261 aaaatccgtt aaagagttac tagggatcac aattatggaa agaagttcct ttgaaaaaaa
     4321 tccgattgac tttttagaag ctaaaggata taaggaagtt aaaaaagact taatcattaa
     4381 actacctaaa tatagtcttt ttgagttaga aaacggtcgt aaacggatgc tggctagtgc
     4441 cggagaatta caaaaaggaa atgagctggc tctgccaagc aaatatgtga attttttata
     4501 tttagctagt cattatgaaa agttgaaggg tagtccagaa gataacgaac aaaaacaatt
     4561 gtttgtggag cagcataagc attatttaga tgagattatt gagcaaatca gtgaattttc
     4621 taagcgtgtt attttagcag atgccaattt agataaagtt cttagtgcat ataacaaaca
     4681 tagagacaaa ccaatacgtg aacaagcaga aaatattatt catttattta cgttgacgaa
     4741 tcttggagct cccgctgctt ttaaatattt tgatacaaca attgatcgta aacgatatac
     4801 gtctacaaaa gaagttttag atgccactct tatccatcaa tccatcactg gtctttatga
     4861 aacacgcatt gatttgagtc agctaggagg tgactaactc gagtaaggat ctccaggcat
     4921 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg ttgtttgtcg
     4981 gtgaacgctc tctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa
     5041 acgggtcttg aggggttttt tgctgaaacc tcaggcattt gagaagcaca cggtcacact
     5101 gcttccggta gtcaataaac cggtaaacca gcaatagaca taagcggcta tttaacgacc
     5161 ctgccctgaa ccgacgaccg ggtcatcgtg gccggatctt gcggcccctc ggcttgaacg
     5221 aattgttaga cattatttgc cgactacctt ggtgatctcg cctttcacgt agtggacaaa
     5281 ttcttccaac tgatctgcgc gcgaggccaa gcgatcttct tcttgtccaa gataagcctg
     5341 tctagcttca agtatgacgg gctgatactg ggccggcagg cgctccattg cccagtcggc
     5401 agcgacatcc ttcggcgcga ttttgccggt tactgcgctg taccaaatgc gggacaacgt
     5461 aagcactaca tttcgctcat cgccagccca gtcgggcggc gagttccata gcgttaaggt
     5521 ttcatttagc gcctcaaata gatcctgttc aggaaccgga tcaaagagtt cctccgccgc
     5581 tggacctacc aaggcaacgc tatgttctct tgcttttgtc agcaagatag ccagatcaat
     5641 gtcgatcgtg gctggctcga agatacctgc aagaatgtca ttgcgctgcc attctccaaa
     5701 ttgcagttcg cgcttagctg gataacgcca cggaatgatg tcgtcgtgca caacaatggt
     5761 gacttctaca gcgcggagaa tctcgctctc tccaggggaa gccgaagttt ccaaaaggtc
     5821 gttgatcaaa gctcgccgcg ttgtttcatc aagccttacg gtcaccgtaa ccagcaaatc
     5881 aatatcactg tgtggcttca ggccgccatc cactgcggag ccgtacaaat gtacggccag
     5941 caacgtcggt tcgagatggc gctcgatgac gccaactacc tctgatagtt gagtcgatac
     6001 ttcggcgatc accgcttccc tcatactctt cctttttcaa tattattgaa gcatttatca
     6061 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagc
     6121 tagctcactc ggtcgctacg ctccgggcgt gagactgcgg cgggcgctgc ggacacatac
     6181 aaagttaccc acagattccg tggataagca ggggactaac atgtgaggca aaacagcagg
     6241 gccgcgccgg tggcgttttt ccataggctc cgccctcctg ccagagttca cataaacaga
     6301 cgcttttccg gtgcatctgt gggagccgtg aggctcaacc atgaatctga cagtacgggc
     6361 gaaacccgac aggacttaaa gatccccacc gtttccggcg ggtcgctccc tcttgcgctc
     6421 tcctgttccg accctgccgt ttaccggata cctgttccgc ctttctccct tacgggaagt
     6481 gtggcgcttt ctcatagctc acacactggt atctcggctc ggtgtaggtc gttcgctcca
     6541 agctgggctg taagcaagaa ctccccgttc agcccgactg ctgcgcctta tccggtaact
     6601 gttcacttga gtccaacccg gaaaagcacg gtaaaacgcc actggcagca gccattggta
     6661 actgggagtt cgcagaggat ttgtttagct aaacacgcgg ttgctcttga agtgtgcgcc
     6721 aaagtccggc tacactggaa ggacagattt ggttgctgtg ctctgcgaaa gccagttacc
     6781 acggttaagc agttccccaa ctgacttaac cttcgatcaa accacctccc caggtggttt
     6841 tttcgtttac agggcaaaag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6901 tcttttctac tgaaccgctc tagcactagt caaaaaaagc accgactcgg tgccactttt
     6961 tcaagttgat aacggactag ccttatttta acttgctatt tctagctcta aaacctggta
     7021 acaggattag cagatgtgtg aaattgttat ccgctcacaa ttccacacat tatacgagcc
     7081 ggatgattaa ttgtcaacag ctcatttcag aatatttgcc agaaccgtta tgatgtcggc
     7141 gcaaaaaaca ttatccagaa cgggagtgcg ccttgagcga cacgaattat gcagtgattt
     7201 acgacctgca cagccatacc acagcttccg atggctgcct gacgccagaa gcattggtgc
     7261 accgtgcagt cgatgataag ctgtcaaacc agatcaattc gcgctaactc acattaattg
     7321 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     7381 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc
     7441 accagtgaga cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc
     7501 aagcggtcca cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttgacggc
     7561 gggatataac atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca
     7621 acgcgcagcc cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca
     7681 accagcatcg cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg
     7741 gacatggcac tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga
     7801 tatttatgcc agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac
     7861 agcgcgattt gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct
     7921 tcatgggaga aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc
     7981 ggaacattag tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta
     8041 atgatcagcc cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg
     8101 acgccgcttc gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat
     8161 ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca
     8221 atcagcaacg actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc
     8281 tccgccatcg ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc
     8341 accacgcggg aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt
     8401 actggtttca cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg
     8461 cgaaaggttt tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgact
//
