LOCUS       40924_3571        5247 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pAC-crRNA-Cm, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5247)
  AUTHORS   Yan MY, Yan HQ, Ren GX, Zhao JP, Guo XP, Sun YC.
  TITLE     CRISPR-Cas12a-assisted recombineering in bacteria
  JOURNAL   Appl. Environ. Microbiol. (2017) In press
  PUBMED    28646112
REFERENCE   2  (bases 1 to 5247)
  AUTHORS   Yan M-Y., Yan H-Q., Ren G-X., Zhao J-P., Guo X-P., Sun Y-C.
  TITLE     Direct Submission
  JOURNAL   Submitted (08-JUN-2017) Moh Key Laboratory of Systems Biology of 
            Pathogens, Chinese Academy of Medical Sciences and Peking Union 
            Medical College Institute of Pathogen Biology, 9, Dongdan Santiao, 
            Dongcheng District, Beijing 100730, China
REFERENCE   3  (bases 1 to 5247)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5247)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Appl. 
            Environ. Microbiol. (2017) In press"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (08-JUN-2017) Moh Key Laboratory of Systems Biology of Pathogens, 
            Chinese Academy of Medical Sciences and Peking Union Medical College
            Institute of Pathogen Biology, 9, Dongdan Santiao, Dongcheng 
            District, Beijing 100730, China"
COMMENT     SGRef: number: 3; type: "Journal Article"
COMMENT     ##Assembly-Data-START##
            Sequencing Technology :: Sanger dideoxy sequencing 
            ##Assembly-Data-END##
FEATURES             Location/Qualifiers
     source          1..5247
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(4..660)
                     /codon_start=1
                     /label=CmR
                     /note="chloramphenicol acetyltransferase"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     promoter        complement(661..763)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      complement(1289..1834)
                     /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."
     promoter        1946..1974
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     misc_feature    2003..2038
                     /label=Direct Repeat
                     /note="Direct Repeat"
     CDS             2058..2774
                     /codon_start=1
                     /label=AcGFP1
                     /note="Aequorea coerulescens GFP"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     misc_feature    2798..2833
                     /label=Direct Repeat
                     /note="Direct Repeat"
     promoter        2937..3382
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             3383..4801
                     /codon_start=1
                     /label=SacB
                     /note="secreted levansucrase that renders bacterial growth 
                     sensitive to sucrose"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
ORIGIN
        1 ttacgccccg ccctgccact catcgcagta ctgttgtaat tcattaagca ttctgccgac
       61 atggaagcca tcacagacgg catgatgaac ctgaatcgcc agcggcatca gcaccttgtc
      121 gccttgcgta taatatttgc ccatggtgaa aacgggggcg aagaagttgt ccatattggc
      181 cacgtttaaa tcaaaactgg tgaaactcac ccagggattg gctgagacga aaaacatatt
      241 ctcaataaac cctttaggga aataggccag gttttcaccg taacacgcca catcttgcga
      301 atatatgtgt agaaactgcc ggaaatcgtc gtggtattca ctccaaagcg atgaaaacgt
      361 ttcagtttgc tcatggaaaa cggtgtaaca agggtgaaca ctatcccata tcaccagctc
      421 accgtctttc attgccatac ggaattccgg atgagcattc atcaggcggg caagaatgtg
      481 aataaaggcc ggataaaact tgtgcttatt tttctttacg gtctttaaaa aggccgtaat
      541 atccagctga acggtctggt tataggtaca ttgagcaact gactgaaatg cctcaaaatg
      601 ttctttacga tgccattggg atatatcaac ggtggtatat ccagtgattt ttttctccat
      661 tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg gtagtgatct
      721 tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc attttcgcca
      781 aaagttggcc cagggcttcc cggtatcaac agggacacca ggatttattt attctgcgaa
      841 gtgatcttcc gtcacaggta tttattcggc gcaaagtgcg tcgggtgatg ctgccaactt
      901 actgatttag tgtatgatgg tgtttttgag gtgcttcagt ggcttctgtt tctatcagct
      961 gtccctcctg ttcagctact gacggggtgg tgcgtaacgg caaaagcacc gccggacatc
     1021 agcgctagcg gagtgtatac tggcttacta tgttggcact gatgagggtg tcagtgaagt
     1081 gcttcatgtg gcaggagaaa aaaggctgca ccggtgcgtc agcagaatat gtgatacagg
     1141 atatattccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact gcggcgagcg
     1201 gaaatggctt acgaacgggg cggagatttc ctggaagatg ccaggaagat acttaacagg
     1261 gaagtgagag ggccgcggca aagccgtttt tccataggct ccgcccccct gacaagcatc
     1321 acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa agataccagg
     1381 cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg tttaccggtg
     1441 tcattccgct gttatggccg cgtttgtctc attccacgcc tgacactcag ttccgggtag
     1501 gcagttcgct ccaagctgga ctgtatgcac gaaccccccg ttcagtccga ccgctgcgcc
     1561 ttatccggta actatcgtct tgagtccaac ccggaaagac atgcaaaagc accactggca
     1621 gcagccactg gtaattgatt tagaggagtt agtcttgaag tcatgcgccg gttaaggcta
     1681 aactgaaagg acaagttttg gtgactgcgc tcctccaagc cagttacctc ggttcaaaga
     1741 gttggtagct cagagaacct tcgaaaaacc gccctgcaag gcggtttttt cgttttcaga
     1801 gcaagagatt acgcgcagac caaaacgatc tcaagaagat catcttatta atcagataaa
     1861 atatttctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag tcagccccat
     1921 acgatataag ttgtaattct catgtttgac agcttatcat cgataagctt taatgcggta
     1981 gtttatcaca gttaaattgc tagtctaaga actttaaata atttctactg ttgtagatga
     2041 gacctttcct gctccagatg gtgagcaagg gcgccgagct gttcaccggc atcgtgccca
     2101 tcctgatcga gctgaatggc gatgtgaatg gccacaagtt cagcgtgagc ggcgagggcg
     2161 agggcgatgc cacctacggc aagctgaccc tgaagttcat ctgcaccacc ggcaagctgc
     2221 ctgtgccctg gcccaccctg gtgaccaccc tgagctacgg cgtgcagtgc ttctcacgct
     2281 accccgatca catgaagcag cacgacttct tcaagagcgc catgcctgag ggctacatcc
     2341 aggagcgcac catcttcttc gaggatgacg gcaactacaa gtcgcgcgcc gaggtgaagt
     2401 tcgagggcga taccctggtg aatcgcatcg agctgaccgg caccgatttc aaggaggatg
     2461 gcaacatcct gggcaataag atggagtaca actacaacgc ccacaatgtg tacatcatga
     2521 ccgacaaggc caagaatggc atcaaggtga acttcaagat ccgccacaac atcgaggatg
     2581 gcagcgtgca gctggccgac cactaccagc agaatacccc catcggcgat ggccctgtgc
     2641 tgctgcccga taaccactac ctgtccaccc agagcgccct gtccaaggac cccaacgaga
     2701 agcgcgatca catgatctac ttcggcttcg tgaccgccgc cgccatcacc cacggcatgg
     2761 atgagctgta caagtgactg gagtgtgaaa ggtctctgtc taagaacttt aaataatttc
     2821 tactgttgta gatgtcagtt tcacctgttt tacgtaaaaa cccgcttcgg cgggttttta
     2881 cttttggctc gagcattcaa atatgtatcc gctcaccgcg atccttttta acccatcaca
     2941 tatacctgcc gttcactatt atttagtgaa atgagatatt atgatatttt ctgaattgtg
     3001 attaaaaagg caactttatg cccatgcaac agaaactata aaaaatacag agaatgaaaa
     3061 gaaacagata gattttttag ttctttaggc ccgtagtctg caaatccttt tatgattttc
     3121 tatcaaacaa aagaggaaaa tagaccagtt gcaatccaaa cgagagtcta atagaatgag
     3181 gtcgaaaagt aaatcgcgcg ggtttgttac tgataaagca ggcaagacct aaaatgtgta
     3241 aagggcaaag tgtatacttt ggcgtcaccc cttacatatt ttaggtcttt ttttattgtg
     3301 cgtaactaac ttgccatctt caaacaggag ggctggaaga agcagaccgc taacacagta
     3361 cataaaaaag gagacatgaa cgatgaacat caaaaagttt gcaaaacaag caacagtatt
     3421 aacctttact accgcactgc tggcaggagg cgcaactcaa gcgtttgcga aagaaacgaa
     3481 ccaaaagcca tataaggaaa catacggcat ttcccatatt acacgccatg atatgctgca
     3541 aatccctgaa cagcaaaaaa atgaaaaata tcaagttcct gaattcgatt cgtccacaat
     3601 taaaaatatc tcttctgcaa aaggcctgga cgtttgggac agctggccat tacaaaacgc
     3661 tgacggcact gtcgcaaact atcacggcta ccacatcgtc tttgcattag ccggagatcc
     3721 taaaaatgcg gatgacacat cgatttacat gttctatcaa aaagtcggcg aaacttctat
     3781 tgacagctgg aaaaacgctg gccgcgtctt taaagacagc gacaaattcg atgcaaatga
     3841 ttctatccta aaagaccaaa cacaagaatg gtcaggttca gccacattta catctgacgg
     3901 aaaaatccgt ttattctaca ctgatttctc cggtaaacat tacggcaaac aaacactgac
     3961 aactgcacaa gttaacgtat cagcatcaga cagctctttg aacatcaacg gtgtagagga
     4021 ttataaatca atctttgacg gtgacggaaa aacgtatcaa aatgtacagc agttcatcga
     4081 tgaaggcaac tacagctcag gcgacaacca tacgctgaga gatcctcact acgtagaaga
     4141 taaaggccac aaatacttag tatttgaagc aaacactgga actgaagatg gctaccaagg
     4201 cgaagaatct ttatttaaca aagcatacta tggcaaaagc acatcattct tccgtcaaga
     4261 aagtcaaaaa cttctgcaaa gcgataaaaa acgcacggct gagttagcaa acggcgctct
     4321 cggtatgatt gagctaaacg atgattacac actgaaaaaa gtgatgaaac cgctgattgc
     4381 atctaacaca gtaacagatg aaattgaacg cgcgaacgtc tttaaaatga acggcaaatg
     4441 gtacctgttc actgactccc gcggatcaaa aatgacgatt gacggcatta cgtctaacga
     4501 tatttacatg cttggttatg tttctaattc tttaactggc ccatacaagc cgctgaacaa
     4561 aactggcctt gtgttaaaaa tggatcttga tcctaacgat gtaaccttta cttactcaca
     4621 cttcgctgta cctcaagcga aaggaaacaa tgtcgtgatt acaagctata tgacaaacag
     4681 aggattctac gcagacaaac aatcaacgtt tgcgccaagc ttcctgctga acatcaaagg
     4741 caagaaaaca tctgttgtca aagacagcat ccttgaacaa ggacaattaa cagttaacaa
     4801 ataaaaacgc aaaagaaaat gccgatnncc ggtttattga ctaccggaag cagtgtgacc
     4861 gtgtgcttct caaatgcctc aggctgtcta tgtgtgactg ttgagctgta acaagttgtc
     4921 tcaggtgttc aatttcatgt tctagttgct ttgttttact ggtttcacct gttctattag
     4981 gtgttacatg ctgttcatct gttacattgt cgatctgttc atggtgaaca gctttaaatg
     5041 caccaaaaac tcgtaaaagc tctgatgtat ctatcttttt tacaccgttt tcatctgtgc
     5101 atatgataag cggctattta acgaccctgc cctgaaccga cgaccgggtc gaatttgctt
     5161 tcgaatttct gccattcatc cgcttattat cacttattca ggcgtagcac caggcgttta
     5221 agggcaccaa taactgcctt aaaaaaa
//
