pSEVA2514-rec2-mutLE36KPP vector (V016316)

LOCUS       62056_19505        9354 bp DNA     circular SYN 28-MAR-2020
DEFINITION  Cloning vector pSEVA2514-rec2-mutLE36KPP, complete sequence.
ACCESSION   MN180222
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9354)
  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   bioRxivorg (2019)
REFERENCE   2  (bases 1 to 9354)
  AUTHORS   Aparicio T, Martinez-Garcia E, de Lorenzo V.
  TITLE     Direct Submission
  JOURNAL   Submitted (15-JUL-2019) Systems Biology Program, Centro Nacional de 
            Biotecnologia, CSIC, Darwin 3, Madrid, Madrid 28049, Spain
REFERENCE   3  (bases 1 to 9354)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxivorg 
            (2019)"
COMMENT     SGRef: number: 2; type: "Journal Article"; journalName: "Submitted 
            (15-JUL-2019) Systems Biology Program, Centro Nacional de 
            Biotecnologia, CSIC, Darwin 3, Madrid, Madrid 28049, Spain"
FEATURES             Location/Qualifiers
     source          1..9354
                     /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"
                     /label=rec2
                     /note="Rec2 recombinase"
                     /protein_id="QIP75692.1"
                     /translation="MSNAALAERQNETQLLDAPAGAVQATESSMMIQMIQRAAADPAVD
                     VDKMERLMQMHERFVDRQASAAFNAAMVRAQRRIKPVARRALNVQTNSTYARLEDIDRE
                     ISPIFTEEGFSLSFGTGDSHLAGYIRVICDVMHDQGHTRQYKMDLPLDATGIGGKTNKT
                     GVHAHGSTNSYARRYLTMNIFNVVMANEDTDGNAEPPEEPVITSRQAAQLEALLKKCSP
                     TMAGLFIEKYGCASNVYKSEFDEVLAKLTRSANRTQQEPVDANHH"
     gene            2287..4185
                     /gene="mutLE36KPP"
                     /label=mutLE36KPP
     CDS             2287..4185
                     /codon_start=1
                     /transl_table=11
                     /gene="mutLE36KPP"
                     /product="MutLE36KPP"
                     /label=mutLE36KPP
                     /note="Dominant negative allele"
                     /protein_id="QIP75693.1"
                     /translation="MSGGSRIQLLSPRLANQIAAGEVVERPASVAKELLKNSLDSGARR
                     IDIEVEQGGVKLLKVRDDGSGISADDLPLALARHATSKIRELEDLEGVLSLGFRGEALA
                     SISSVARLTLTSRTASASEAWQVETEGRDMTPRVQPAAHPVGTSVEVRDLFFNTPARRK
                     FLKAEKTEFDHLQEVIRRLALARFDVGFHLRHNGKSILSLHEAHDEIARARRVGAICGP
                     GFMEQALPIDVERNGLRLWGWVGLPTFSRSQADLQYFFVNGRAVRDKLVAHAVRQAYRD
                     VLFNGRHPTFVLFLELEPNGVDVNVHPTKHEVRFREGRSVHDFLYGTLHRALADVRPED
                     QLAAPAAVPELVRPTGQQAGEFGPQGEMRLASPVLEQPRAPQQSFSNGGSGAGYQYQYT
                     PRPSQPLPAAEAQAVYREFYKPLETGAAPATALPESQGDIPPLGYALAQLKGIYILAEN
                     AVGLVLVDMHAAHERIMYERLKVAMASEGLSGQPLLVPETLALSQREADCAEEHAQWFQ
                     RLGFELQRLGPETLAIRQIPALLKQAEANRLVQDVLADLMEYGTSDRIQAHLNELLGTM
                     ACHGAVRANRRLAIPEMNALLRDMENTERSGQCNHGRPTWTQMGLDDLDKLFLRGR"
     primer_bind     complement(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(5564..6412)
                     /label=RSF1010 RepC
                     /note="replication protein C of the broad-host-range
                     plasmid RSF1010 (Scholz et al., 1989)"
     CDS             complement(6402..7238)
                     /label=RSF1010 RepA
                     /note="replication protein A of the broad-host-range
                     plasmid RSF1010 (Scholz et al., 1989)"
     CDS             complement(7752..8720)
                     /label=RSF1010 RepB
                     /note="replication protein B of the broad-host-range
                     plasmid RSF1010 (Scholz et al., 1989)"
     promoter        complement(8744..8774)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an 'up' mutation"
     rep_origin      8830..9224
                     /label=RSF1010 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     RSF1010; requires the RSF1010 RepA/B/C proteins for 
                     replication (Scholz et al., 1989)"
     terminator      complement(9262..9348)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"