pSEVA2214-rec2-mutLE36KPP vector (V015710)

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LOCUS       62056_19475        7902 bp DNA     circular SYN 04-MAR-2020
DEFINITION  Expression vector pSEVA2214-rec2-mutLE36KPP, complete sequence.
ACCESSION   MN688223
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7902)
  AUTHORS   Aparicio T, Nyerges A, Martinez-Garcia E, de Lorenzo V.
  TITLE     High-efficiency multi-site genomic editing (HEMSE) of Pseudomonas 
            putida through thermoinducible ssDNA recombineering
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 7902)
  AUTHORS   Aparicio T, Nyerges A, Martinez-Garcia E, de Lorenzo V.
  TITLE     Direct Submission
  JOURNAL   Submitted (12-NOV-2019) Systems Biology Program, Centro Nacional de 
            Biotecnologia, CSIC, Darwin 3, Madrid, Madrid 28049, Spain
REFERENCE   3  (bases 1 to 7902)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (12-NOV-2019) Systems Biology Program, Centro Nacional de 
            Biotecnologia, CSIC, Darwin 3, Madrid, Madrid 28049, Spain"
FEATURES             Location/Qualifiers
     source          1..7902
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(12..722)
                     /label=lambda repressor (ts)
                     /note="temperature-sensitive variant of the phage lambda
                     repressor"
     regulatory      complement(729..752)
                     /label=tir region
                     /note="tir region"
                     /regulatory_class="other"
     regulatory      complement(753..858)
                     /label=PKan (Km promoter)
                     /note="PKan (Km promoter)"
                     /regulatory_class="promoter"
     primer_bind     1009..1032
                     /label=R24
                     /note="R24"
     regulatory      1033..1414
                     /label=PL promoter
                     /note="PL promoter"
                     /regulatory_class="promoter"
     gene            1455..2258
                     /gene="rec2"
                     /label=rec2
     CDS             1455..2258
                     /codon_start=1
                     /transl_table=11
                     /gene="rec2"
                     /product="Rec2 recombinase"
                     /label=rec2
                     /protein_id="QIE07183.1"
                     /translation="MSNAALAERQNETQLLDAPAGAVQATESSMMIQMIQRAAADPAVD
                     VDKMERLMQMHERFVDRQASAAFNAAMVRAQRRIKPVARRALNVQTNSTYARLEDIDRE
                     ISPIFTEEGFSLSFGTGDSHLAGYIRVICDVMHDQGHTRQYKMDLPLDATGIGGKTNKT
                     GVHAHGSTNSYARRYLTMNIFNVVMANEDTDGNAEPPEEPVITSRQAAQLEALLKKCSP
                     TMAGLFIEKYGCASNVYKSEFDEVLAKLTRSANRTQQEPVDANHH"
     gene            2287..4185
                     /gene="mutL"
                     /allele="mutLE36KPP"
                     /label=mutL
     CDS             2287..4185
                     /codon_start=1
                     /transl_table=11
                     /gene="mutL"
                     /allele="mutLE36KPP"
                     /product="MutLE36KPP"
                     /label=mutL
                     /note="MutLE36KPP; dominant negative allele of MMR system"
                     /protein_id="QIE07184.1"
                     /translation="MSGGSRIQLLSPRLANQIAAGEVVERPASVAKELLKNSLDSGARR
                     IDIEVEQGGVKLLKVRDDGSGISADDLPLALARHATSKIRELEDLEGVLSLGFRGEALA
                     SISSVARLTLTSRTASASEAWQVETEGRDMTPRVQPAAHPVGTSVEVRDLFFNTPARRK
                     FLKAEKTEFDHLQEVIRRLALARFDVGFHLRHNGKSILSLHEAHDEIARARRVGAICGP
                     GFMEQALPIDVERNGLRLWGWVGLPTFSRSQADLQYFFVNGRAVRDKLVAHAVRQAYRD
                     VLFNGRHPTFVLFLELEPNGVDVNVHPTKHEVRFREGRSVHDFLYGTLHRALADVRPED
                     QLAAPAAVPELVRPTGQQAGEFGPQGEMRLASPVLEQPRAPQQSFSNGGSGAGYQYQYT
                     PRPSQPLPAAEAQAVYREFYKPLETGAAPATALPESQGDIPPLGYALAQLKGIYILAEN
                     AVGLVLVDMHAAHERIMYERLKVAMASEGLSGQPLLVPETLALSQREADCAEEHAQWFQ
                     RLGFELQRLGPETLAIRQIPALLKQAEANRLVQDVLADLMEYGTSDRIQAHLNELLGTM
                     ACHGAVRANRRLAIPEMNALLRDMENTERSGQCNHGRPTWTQMGLDDLDKLFLRGR"
     primer_bind     4200..4223
                     /label=F24
                     /note="F24"
     terminator      4234..4328
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             4460..5272
                     /label=KanR
                     /note="aminoglycoside phosphotransferase"
     oriT            5442..5550
                     /label=oriT
                     /note="incP origin of transfer"
     CDS             complement(5614..6759)
                     /label=trfA
                     /note="trans-acting replication protein that binds to and 
                     activates oriV"
     rep_origin      complement(7152..7683)
                     /direction=LEFT
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(7810..7896)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"