LOCUS       Exported                7795 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Multigene editing in the Escherichia coli genome using the 
            CRISPR-Cas9 system.
ACCESSION   .
VERSION     .
KEYWORDS    pSU-araC-Cas9
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7795)
  AUTHORS   Jiang Y, Chen B, Duan C, Sun B, Yang J, Yang S
  TITLE     Multigene editing in the Escherichia coli genome using the 
            CRISPR-Cas9 system.
  JOURNAL   Appl Environ Microbiol. 2015 Jan 30. pii: AEM.04023-14.
  PUBMED    25636838
REFERENCE   2  (bases 1 to 7795)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7795
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     134..153
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(220..322)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     709..731
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     723..740
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     724..740
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             complement(855..1733)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        1760..2044
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     1967..1986
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     RBS             2071..2079
                     /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             2088..6194
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKK
                     NLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEES
                     FLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIK
                     FRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRL
                     ENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQ
                     IGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVR
                     QQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL
                     RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARG
                     NSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEY
                     FTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFD
                     SVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEER
                     LKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFM
                     QLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGR
                     HKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLY
                     LYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVP
                     SEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKH
                     VAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYL
                     NAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKT
                     EITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSK
                     ESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI
                     TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE
                     LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD
                     ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL
                     DATLIHQSITGLYETRIDLSQLGGD"
     primer_bind     complement(6266..6282)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6266..6282)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6279..6301)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6290..6306
                     /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)."
     promoter        complement(6314..6344)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6359..6380
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6615..6632)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6749..7294)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(6829..6848)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7355..219)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
ORIGIN
        1 gaattccgga tgagcattca tcaggcgggc aagaatgtga ataaaggccg gataaaactt
       61 gtgcttattt ttctttacgg tctttaaaaa ggccgtaata tccagctgaa cggtctggtt
      121 ataggtacat tgagcaactg actgaaatgc ctcaaaatgt tctttacgat gccattggga
      181 tatatcaacg gtggtatatc cagtgatttt tttctccatt ttagcttcct tagctcctga
      241 aaatctcgat aactcaaaaa atacgcccgg tagtgatctt atttcattat ggtgaaagtt
      301 ggaacctctt acgtgccgat caacgtctca ttttcgccaa aagttggccc agggcttccc
      361 ggtatcaaca gggacaccag gatttattta ttctgcgaag tgatcttccg tcacaggtat
      421 ttattcggcg caaagtgcgt cgggtgatgc tgccaactta ctgatttagt gtatgatggt
      481 gtttttgagg tgctccagtg gcttctgttt ctatcagctg tccctcctgt tcagctactg
      541 acggggtggt gcgtaacggc aaaagcaccg ccggacatca gcgccattcg ccattcaggc
      601 tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctggcga
      661 aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc cagtcacgac
      721 gttgtaaaac gacggccagt gccaagcttg catgcctgca gcatgagcgg atacatattt
      781 gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca
      841 cctgcatcga tttattatga caacttgacg gctacatcat tcactttttc ttcacaaccg
      901 gcacggaact cgctcgggct ggccccggtg cattttttaa atacccgcga gaagtagagt
      961 tgatcgtcaa aaccaacatt gcgaccgacg gtggcgatag gcatccgggt ggtgctcaaa
     1021 agcagcttcg cctggctgat acgttggtcc tcgcgccagc ttaagacgct aatccctaac
     1081 tgctggcgga aaagatgtga cagacgcgac ggcgacaagc aaacatgctg tgcgacgctg
     1141 gcgatatcaa aattgctgtc tgccaggtga tcgctgatgt actgacaagc ctcgcgtacc
     1201 cgattatcca tcggtggatg gagcgactcg ttaatcgctt ccatgcgccg cagtaacaat
     1261 tgctcaagca gatttatcgc cagcagctcc gaatagcgcc cttccccttg cccggcgtta
     1321 atgatttgcc caaacaggtc gctgaaatgc ggctggtgcg cttcatccgg gcgaaagaac
     1381 cccgtattgg caaatattga cggccagtta agccattcat gccagtaggc gcgcggacga
     1441 aagtaaaccc actggtgata ccattcgcga gcctccggat gacgaccgta gtgatgaatc
     1501 tctcctggcg ggaacagcaa aatatcaccc ggtcggcaaa caaattctcg tccctgattt
     1561 ttcaccaccc cctgaccgcg aatggtgaga ttgagaatat aacctttcat tcccagcggt
     1621 cggtcgataa aaaaatcgag ataaccgttg gcctcaatcg gcgttaaacc cgccaccaga
     1681 tgggcattaa acgagtatcc cggcagcagg ggatcatttt gcgcttcagc catacttttc
     1741 atactcccgc cattcagaga agaaaccaat tgtccatatt gcatcagaca ttgccgtcac
     1801 tgcgtctttt actggctctt ctcgctaacc aaaccggtaa ccccgcttat taaaagcatt
     1861 ctgtaacaaa gcgggaccaa agccatgaca aaaacgcgta acaaaagtgt ctataatcac
     1921 ggcagaaaag tccacattga ttatttgcac ggcgtcacac tttgctatgc catagcattt
     1981 ttatccataa gattagcgga tcctacctga cgctttttat cgcaactctc tactgtttct
     2041 ccatacccgt ttttttggga attcgagctc taaggaggtt ataaaaaatg gataagaaat
     2101 actcaatagg cttagatatc ggcacaaata gcgtcggatg ggcggtgatc actgatgaat
     2161 ataaggttcc gtctaaaaag ttcaaggttc tgggaaatac agaccgccac agtatcaaaa
     2221 aaaatcttat aggggctctt ttatttgaca gtggagagac agcggaagcg actcgtctca
     2281 aacggacagc tcgtagaagg tatacacgtc ggaagaatcg tatttgttat ctacaggaga
     2341 ttttttcaaa tgagatggcg aaagtagatg atagtttctt tcatcgactt gaagagtctt
     2401 ttttggtgga agaagacaag aagcatgaac gtcatcctat ttttggaaat atagtagatg
     2461 aagttgctta tcatgagaaa tatccaacta tctatcatct gcgaaaaaaa ttggtagatt
     2521 ctactgataa agcggatttg cgcttaatct atttggcctt agcgcatatg attaagtttc
     2581 gtggtcattt tttgattgag ggagatttaa atcctgataa tagtgatgtg gacaaactat
     2641 ttatccagtt ggtacaaacc tacaatcaat tatttgaaga aaaccctatt aacgcaagtg
     2701 gagtagatgc taaagcgatt ctttctgcac gattgagtaa atcaagacga ttagaaaatc
     2761 tcattgctca gctccccggt gagaagaaaa atggcttatt tgggaatctc attgctttgt
     2821 cattgggttt gacccctaat tttaaatcaa attttgattt ggcagaagat gctaaattac
     2881 agctttcaaa agatacttac gatgatgatt tagataattt attggcgcaa attggagatc
     2941 aatatgctga tttgtttttg gcagctaaga atttatcaga tgctatttta ctttcagata
     3001 tcctaagagt aaatactgaa ataactaagg ctcccctatc agcttcaatg attaaacgct
     3061 acgatgaaca tcatcaagac ttgactcttt taaaagcttt agttcgacaa caacttccag
     3121 aaaagtataa agaaatcttt tttgatcaat caaaaaacgg atatgcaggt tatattgatg
     3181 ggggagctag ccaagaagaa ttttataaat ttatcaaacc aattttagaa aaaatggatg
     3241 gtactgagga attattggtg aaactaaatc gtgaagattt gctgcgcaag caacggacct
     3301 ttgacaacgg ctctattccc catcaaattc acttgggtga gctgcatgct attttgagaa
     3361 gacaagaaga cttttatcca tttttaaaag acaatcgtga gaagattgaa aaaatcttga
     3421 cttttcgaat tccttattat gttggtccat tggcgcgtgg caatagtcgt tttgcatgga
     3481 tgactcggaa gtctgaagaa acaattaccc catggaattt tgaagaagtt gtcgataaag
     3541 gtgcttcagc tcaatcattt attgaacgca tgacaaactt tgataaaaat cttccaaatg
     3601 aaaaagtact accaaaacat agtttgcttt atgagtattt tacggtttat aacgaattga
     3661 caaaggtcaa atatgttact gaaggaatgc gaaaaccagc atttctttca ggtgaacaga
     3721 agaaagccat tgttgattta ctcttcaaaa caaatcgaaa agtaaccgtt aagcaattaa
     3781 aagaagatta tttcaaaaaa atagaatgtt ttgatagtgt tgaaatttca ggagttgaag
     3841 atagatttaa tgcttcatta ggtacctacc atgatttgct aaaaattatt aaagataaag
     3901 attttttgga taatgaagaa aatgaagata tcttagagga tattgtttta acattgacct
     3961 tatttgaaga tagggagatg attgaggaaa gacttaaaac atatgctcac ctctttgatg
     4021 ataaggtgat gaaacagctt aaacgtcgcc gttatactgg ttggggacgt ttgtctcgaa
     4081 aattgattaa tggtattagg gataagcaat ctggcaaaac aatattagat tttttgaaat
     4141 cagatggttt tgccaatcgc aattttatgc agctgatcca tgatgatagt ttgacattta
     4201 aagaagacat tcaaaaagca caagtgtctg gacaaggcga tagtttacat gaacatattg
     4261 caaatttagc tggtagccct gctattaaaa aaggtatttt acagactgta aaagttgttg
     4321 atgaattggt caaagtaatg gggcggcata agccagaaaa tatcgttatt gaaatggcac
     4381 gtgaaaatca gacaactcaa aagggccaga aaaattcgcg agagcgtatg aaacgaatcg
     4441 aagaaggtat caaagaatta ggaagtcaga ttcttaaaga gcatcctgtt gaaaatactc
     4501 aattgcaaaa tgaaaagctc tatctctatt atctccaaaa tggaagagac atgtatgtgg
     4561 accaagaatt agatattaat cgtttaagtg attatgatgt cgatcacatt gttccacaaa
     4621 gtttccttaa agacgattca atagacaata aggtcttaac gcgttctgat aaaaatcgtg
     4681 gtaaatcgga taacgttcca agtgaagaag tagtcaaaaa gatgaaaaac tattggagac
     4741 aacttctaaa cgccaagtta atcactcaac gtaagtttga taatttaacg aaagctgaac
     4801 gtggaggttt gagtgaactt gataaagctg gttttatcaa acgccaattg gttgaaactc
     4861 gccaaatcac taagcatgtg gcacaaattt tggatagtcg catgaatact aaatacgatg
     4921 aaaatgataa acttattcga gaggttaaag tgattacctt aaaatctaaa ttagtttctg
     4981 acttccgaaa agatttccaa ttctataaag tacgtgagat taacaattac catcatgccc
     5041 atgatgcgta tctaaatgcc gtcgttggaa ctgctttgat taagaaatat ccaaaacttg
     5101 aatcggagtt tgtctatggt gattataaag tttatgatgt tcgtaaaatg attgctaagt
     5161 ctgagcaaga aataggcaaa gcaaccgcaa aatatttctt ttactctaat atcatgaact
     5221 tcttcaaaac agaaattaca cttgcaaatg gagagattcg caaacgccct ctaatcgaaa
     5281 ctaatgggga aactggagaa attgtctggg ataaagggcg agattttgcc acagtgcgca
     5341 aagtattgtc catgccccaa gtcaatattg tcaagaaaac agaagtacag acaggcggat
     5401 tctccaagga gtcaatttta ccaaaaagaa attcggacaa gcttattgct cgtaaaaaag
     5461 actgggatcc aaaaaaatat ggtggttttg atagtccaac ggtagcttat tcagtcctag
     5521 tggttgctaa ggtggaaaaa gggaaatcga agaagttaaa atccgttaaa gagttactag
     5581 ggatcacaat tatggaaaga agttcctttg aaaaaaatcc gattgacttt ttagaagcta
     5641 aaggatataa ggaagttaaa aaagacttaa tcattaaact acctaaatat agtctttttg
     5701 agttagaaaa cggtcgtaaa cggatgctgg ctagtgccgg agaattacaa aaaggaaatg
     5761 agctggctct gccaagcaaa tatgtgaatt ttttatattt agctagtcat tatgaaaagt
     5821 tgaagggtag tccagaagat aacgaacaaa aacaattgtt tgtggagcag cataagcatt
     5881 atttagatga gattattgag caaatcagtg aattttctaa gcgtgttatt ttagcagatg
     5941 ccaatttaga taaagttctt agtgcatata acaaacatag agacaaacca atacgtgaac
     6001 aagcagaaaa tattattcat ttatttacgt tgacgaatct tggagctccc gctgctttta
     6061 aatattttga tacaacaatt gatcgtaaac gatatacgtc tacaaaagaa gttttagatg
     6121 ccactcttat ccatcaatcc atcactggtc tttatgaaac acgcattgat ttgagtcagc
     6181 taggaggtga ctgaagtata ttttagatga agattatttc ttaatctaga ggatccccgg
     6241 gtaccgagct cgaattcgta atcatgtcat agctgtttcc tgtgtgaaat tgttatccgc
     6301 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     6361 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     6421 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     6481 ggcgctagcg gagtgtatac tggcttacta tgttggcact gatgagggtg tcagtgaagt
     6541 gcttcatgtg gcaggagaaa aaaggctgca ccggtgcgtc agcagaatat gtgatacagg
     6601 atatattccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact gcggcgagcg
     6661 gaaatggctt acgaacgggg cggagatttc ctggaagatg ccaggaagat acttaacagg
     6721 gaagtgagag ggccgcggca aagccgtttt tccataggct ccgcccccct gacaagcatc
     6781 acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa agataccagg
     6841 cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg tttaccggtg
     6901 tcattccgct gttatggccg cgtttgtctc attccacgcc tgacactcag ttccgggtag
     6961 gcagttcgct ccaagctgga ctgtatgcac gaaccccccg ttcagtccga ccgctgcgcc
     7021 ttatccggta actatcgtct tgagtccaac ccggaaagac atgcaaaagc accactggca
     7081 gcagccactg gtaattgatt tagaggagtt agtcttgaag tcatgcgccg gttaaggcta
     7141 aactgaaagg acaagttttg gtgactgcgc tcctccaagc cagttacctc ggttcaaaga
     7201 gttggtagct cagagaacct tcgaaaaacc gccctgcaag gcggtttttt cgttttcaga
     7261 gcaagagatt acgcgcagac caaaacgatc tcaagaagat catcttatta atcagataaa
     7321 atatttctag gcaccaataa ctgccttaaa aaaattacgc cccgccctgc cactcatcgc
     7381 agtactgttg taattcatta agcattctgc cgacatggaa gccatcacaa acggcatgat
     7441 gaacctgaat cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg
     7501 tgaaaacggg ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac
     7561 tcacccaggg attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg
     7621 ccaggttttc accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat
     7681 cgtcgtggta ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt
     7741 aacaagggtg aacactatcc catatcacca gctcaccgtc tttcattgcc atacg
//
