LOCUS       40924_3576        5444 bp DNA     circular SYN 17-DEC-2018
DEFINITION  Expression vector pAC-crRNA-Km, complete sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5444)
  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 5444)
  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 5444)
  TITLE     Direct Submission
REFERENCE   4  (bases 1 to 5444)
  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..5444
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..103)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding
                     chloramphenicol acetyltransferase"
     rep_origin      complement(629..1174)
                     /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        1286..1314
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein
                     gene"
     misc_feature    1343..1378
                     /label=Direct Repeat
                     /note="Direct Repeat"
     CDS             1460..2176
                     /codon_start=1
                     /label=AcGFP1
                     /note="Aequorea coerulescens GFP"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     misc_feature    2200..2235
                     /label=Direct Repeat
                     /note="Direct Repeat"
     promoter        2339..2784
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             2785..4203
                     /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"
     CDS             complement(4653..5444)
                     /codon_start=1
                     /label=NeoR/KanR
                     /note="aminoglycoside phosphotransferase"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
ORIGIN
        1 tttagcttcc ttagctcctg aaaatctcga taactcaaaa aatacgcccg gtagtgatct
       61 tatttcatta tggtgaaagt tggaacctct tacgtgccga tcaacgtctc attttcgcca
      121 aaagttggcc cagggcttcc cggtatcaac agggacacca ggatttattt attctgcgaa
      181 gtgatcttcc gtcacaggta tttattcggc gcaaagtgcg tcgggtgatg ctgccaactt
      241 actgatttag tgtatgatgg tgtttttgag gtgcttcagt ggcttctgtt tctatcagct
      301 gtccctcctg ttcagctact gacggggtgg tgcgtaacgg caaaagcacc gccggacatc
      361 agcgctagcg gagtgtatac tggcttacta tgttggcact gatgagggtg tcagtgaagt
      421 gcttcatgtg gcaggagaaa aaaggctgca ccggtgcgtc agcagaatat gtgatacagg
      481 atatattccg cttcctcgct cactgactcg ctacgctcgg tcgttcgact gcggcgagcg
      541 gaaatggctt acgaacgggg cggagatttc ctggaagatg ccaggaagat acttaacagg
      601 gaagtgagag ggccgcggca aagccgtttt tccataggct ccgcccccct gacaagcatc
      661 acgaaatctg acgctcaaat cagtggtggc gaaacccgac aggactataa agataccagg
      721 cgtttccccc tggcggctcc ctcgtgcgct ctcctgttcc tgcctttcgg tttaccggtg
      781 tcattccgct gttatggccg cgtttgtctc attccacgcc tgacactcag ttccgggtag
      841 gcagttcgct ccaagctgga ctgtatgcac gaaccccccg ttcagtccga ccgctgcgcc
      901 ttatccggta actatcgtct tgagtccaac ccggaaagac atgcaaaagc accactggca
      961 gcagccactg gtaattgatt tagaggagtt agtcttgaag tcatgcgccg gttaaggcta
     1021 aactgaaagg acaagttttg gtgactgcgc tcctccaagc cagttacctc ggttcaaaga
     1081 gttggtagct cagagaacct tcgaaaaacc gccctgcaag gcggtttttt cgttttcaga
     1141 gcaagagatt acgcgcagac caaaacgatc tcaagaagat catcttatta atcagataaa
     1201 atatttctag atttcagtgc aatttatctc ttcaaatgta gcacctgaag tcagccccat
     1261 acgatataag ttgtaattct catgtttgac agcttatcat cgataagctt taatgcggta
     1321 gtttatcaca gttaaattgc tagtctaaga actttaaata atttctactg ttgtagatga
     1381 gacctttcct gctccagttg acggctagct cagtcctagg tacagtgcta gctactagag
     1441 tcacacagga aagtactaga tggtgagcaa gggcgccgag ctgttcaccg gcatcgtgcc
     1501 catcctgatc gagctgaatg gcgatgtgaa tggccacaag ttcagcgtga gcggcgaggg
     1561 cgagggcgat gccacctacg gcaagctgac cctgaagttc atctgcacca ccggcaagct
     1621 gcctgtgccc tggcccaccc tggtgaccac cctgagctac ggcgtgcagt gcttctcacg
     1681 ctaccccgat cacatgaagc agcacgactt cttcaagagc gccatgcctg agggctacat
     1741 ccaggagcgc accatcttct tcgaggatga cggcaactac aagtcgcgcg ccgaggtgaa
     1801 gttcgagggc gataccctgg tgaatcgcat cgagctgacc ggcaccgatt tcaaggagga
     1861 tggcaacatc ctgggcaata agatggagta caactacaac gcccacaatg tgtacatcat
     1921 gaccgacaag gccaagaatg gcatcaaggt gaacttcaag atccgccaca acatcgagga
     1981 tggcagcgtg cagctggccg accactacca gcagaatacc cccatcggcg atggccctgt
     2041 gctgctgccc gataaccact acctgtccac ccagagcgcc ctgtccaagg accccaacga
     2101 gaagcgcgat cacatgatct acttcggctt cgtgaccgcc gccgccatca cccacggcat
     2161 ggatgagctg tacaagtgac tggagtgtga aaggtctctg tctaagaact ttaaataatt
     2221 tctactgttg tagatgtcag tttcacctgt tttacgtaaa aacccgcttc ggcgggtttt
     2281 tacttttggc tcgagcattc aaatatgtat ccgctcaccg cgatcctttt taacccatca
     2341 catatacctg ccgttcacta ttatttagtg aaatgagata ttatgatatt ttctgaattg
     2401 tgattaaaaa ggcaacttta tgcccatgca acagaaacta taaaaaatac agagaatgaa
     2461 aagaaacaga tagatttttt agttctttag gcccgtagtc tgcaaatcct tttatgattt
     2521 tctatcaaac aaaagaggaa aatagaccag ttgcaatcca aacgagagtc taatagaatg
     2581 aggtcgaaaa gtaaatcgcg cgggtttgtt actgataaag caggcaagac ctaaaatgtg
     2641 taaagggcaa agtgtatact ttggcgtcac cccttacata ttttaggtct ttttttattg
     2701 tgcgtaacta acttgccatc ttcaaacagg agggctggaa gaagcagacc gctaacacag
     2761 tacataaaaa aggagacatg aacgatgaac atcaaaaagt ttgcaaaaca agcaacagta
     2821 ttaaccttta ctaccgcact gctggcagga ggcgcaactc aagcgtttgc gaaagaaacg
     2881 aaccaaaagc catataagga aacatacggc atttcccata ttacacgcca tgatatgctg
     2941 caaatccctg aacagcaaaa aaatgaaaaa tatcaagttc ctgaattcga ttcgtccaca
     3001 attaaaaata tctcttctgc aaaaggcctg gacgtttggg acagctggcc attacaaaac
     3061 gctgacggca ctgtcgcaaa ctatcacggc taccacatcg tctttgcatt agccggagat
     3121 cctaaaaatg cggatgacac atcgatttac atgttctatc aaaaagtcgg cgaaacttct
     3181 attgacagct ggaaaaacgc tggccgcgtc tttaaagaca gcgacaaatt cgatgcaaat
     3241 gattctatcc taaaagacca aacacaagaa tggtcaggtt cagccacatt tacatctgac
     3301 ggaaaaatcc gtttattcta cactgatttc tccggtaaac attacggcaa acaaacactg
     3361 acaactgcac aagttaacgt atcagcatca gacagctctt tgaacatcaa cggtgtagag
     3421 gattataaat caatctttga cggtgacgga aaaacgtatc aaaatgtaca gcagttcatc
     3481 gatgaaggca actacagctc aggcgacaac catacgctga gagatcctca ctacgtagaa
     3541 gataaaggcc acaaatactt agtatttgaa gcaaacactg gaactgaaga tggctaccaa
     3601 ggcgaagaat ctttatttaa caaagcatac tatggcaaaa gcacatcatt cttccgtcaa
     3661 gaaagtcaaa aacttctgca aagcgataaa aaacgcacgg ctgagttagc aaacggcgct
     3721 ctcggtatga ttgagctaaa cgatgattac acactgaaaa aagtgatgaa accgctgatt
     3781 gcatctaaca cagtaacaga tgaaattgaa cgcgcgaacg tctttaaaat gaacggcaaa
     3841 tggtacctgt tcactgactc ccgcggatca aaaatgacga ttgacggcat tacgtctaac
     3901 gatatttaca tgcttggtta tgtttctaat tctttaactg gcccatacaa gccgctgaac
     3961 aaaactggcc ttgtgttaaa aatggatctt gatcctaacg atgtaacctt tacttactca
     4021 cacttcgctg tacctcaagc gaaaggaaac aatgtcgtga ttacaagcta tatgacaaac
     4081 agaggattct acgcagacaa acaatcaacg tttgcgccaa gcttcctgct gaacatcaaa
     4141 ggcaagaaaa catctgttgt caaagacagc atccttgaac aaggacaatt aacagttaac
     4201 aaataaaaac gcaaaagaaa atgccgatnn ccggtttatt gactaccgga agcagtgtga
     4261 ccgtgtgctt ctcaaatgcc tcaggctgtc tatgtgtgac tgttgagctg taacaagttg
     4321 tctcaggtgt tcaatttcat gttctagttg ctttgtttta ctggtttcac ctgttctatt
     4381 aggtgttaca tgctgttcat ctgttacatt gtcgatctgt tcatggtgaa cagctttaaa
     4441 tgcaccaaaa actcgtaaaa gctctgatgt atctatcttt tttacaccgt tttcatctgt
     4501 gcatatgata agcggctatt taacgaccct gccctgaacc gacgaccggg tcgaatttgc
     4561 tttcgaattt ctgccattca tccgcttatt atcacttatt caggcgtagc accaggcgtt
     4621 taagggcacc aataactgcc ttaaaaaaat tagaagaact cgtcaagaag gcgatagaag
     4681 gcgatgcgct gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat
     4741 tcgccgccaa gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc
     4801 gccacaccca gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata
     4861 ttcggcaagc aggcatcgcc atgggtcacg acgagatcct cgccgtcggg catgcgcgcc
     4921 ttgagcctgg cgaacagttc ggctggcgcg agcccctgat gctcttcgtc cagatcatcc
     4981 tgatcgacaa gaccggcttc catccgagta cgtgctcgct cgatgcgatg tttcgcttgg
     5041 tggtcgaatg ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc atcagccatg
     5101 atggatactt tctcggcagg agcaaggtga gatgacagga gatcctgccc cggcacttcg
     5161 cccaatagca gccagtccct tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga
     5221 acgcccgtcg tggccagcca cgatagccgc gctgcctcgt cctgcagttc attcagggca
     5281 ccggacaggt cggtcttgac aaaaagaacc gggcgcccct gcgctgacag ccggaacacg
     5341 gcggcatcag agcagccgat tgtctgttgt gcccagtcat agccgaatag cctctccacc
     5401 caagcggccg gagaacctgc gtgcaatcca tcttgttcaa tcat
//
