LOCUS       62056_19500        7202 bp DNA     circular SYN 04-MAR-2020
DEFINITION  Expression vector pSEVA2314-rec2-mutLE36KPP, complete sequence.
ACCESSION   MN688222
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7202)
  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  JOURNAL   Unpublished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 7202)
  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 7202)
  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            "Unpublished"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..7202
                     /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      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="QIE07178.1"
                     /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="MutL"
                     /label=mutL
                     /note="MutLE36KPP; dominant negative allele of MMR system"
                     /protein_id="QIE07179.1"
                     /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(5564..6223)
                     /label=pBBR1 Rep
                     /note="replication protein for the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica"
     rep_origin      complement(6224..6995)
                     /direction=LEFT
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     terminator      complement(7110..7196)
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB
                     gene"
ORIGIN
        1 ttaattaatc agccaaacgt ctcttcaggc cactgactag cgataacttt ccccacaacg
       61 gaacaactct cattgcatgg gatcattggg tactgtgggt ttagtggttg taaaaacacc
      121 tgaccgctat ccctgatcag tttcttgaag gtaaactcat cacccccaag tctggctatg
      181 cagaaatcac ctggctcaac agcctgctca gggtcaacga gaattaacat tccgtcagga
      241 aagctcggct tggagcctgt tggtgcggtc atggaattac cttcaacctc aagccagaat
      301 gcagaatcac tggctttttt ggttgtgctt acccatctct ccgcatcacc tttggtaaag
      361 gttctgagct taggtgagaa catccctgcc tgaacatgag aaaaaacagg gtactcatac
      421 tcacttctaa gtgacggctg catactaacc gcttcataca tctcgtagat ttctctggcg
      481 attgaagggc taaattcttc aacgctaact ttgagaattt ttgtaagcaa tgcggcgtta
      541 taagcattta atgcattgat gccattaaat aaagcaccaa cgcctgactg ccccatcccc
      601 atcttgtctg cgacagattc ctgggataag ccaagttcat ttttcttttt ttcataaatt
      661 gctttaaggc ggcgtgcgtc ctcaagctgc tcttgtgtta atggtttctt ttttgtgctc
      721 atgctagctt tttcctcctt ataaagttaa tcaacacccc ttgtattact gtttatgtaa
      781 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga
      841 ttttgagaca caagacgtgg caaaaaacat tatccagaac gggagtgcgc cttgagcgac
      901 acgaattatg cagtgattta cgacctgcac agccatacca cagcttccga tggctgcctg
      961 acgccagaag cattggtgca ccgtgcagtc gatgataagc tgtcaaacag cggataacaa
     1021 tttcacacag gataacccgg cctcagcgcc gggttttctt tgcctcacga tcgcccccaa
     1081 aacacataac caattgtatt tattgaaaaa taaatagata caactcacta aacatagcaa
     1141 ttcagatctc tcacctacca aacaatgccc ccctgcaaaa aataaattca tataaaaaac
     1201 atacagataa ccatctgcgg tgataaatta tctctggcgg tgttgacata aataccactg
     1261 gcggtgatac tgagcacatc agcaggacgc actgaccacc atgaaggtga cgctcttaaa
     1321 aattaagccc tgaagaaggg cagcattcaa agcagaaggc tttggggtgt gtgatacgaa
     1381 acgaagcatt ggccgtaagt gcgattccgg attacctagg ccgcggccgc gcgaattcag
     1441 aaggagaata taccatgagc aatgcagcct tggccgaacg gcagaatgaa acccagcttc
     1501 tggacgctcc cgccggagcg gtacaagcca ccgaatcgtc catgatgatt caaatgattc
     1561 aacgcgccgc ggctgacccg gcagtggacg tcgacaagat ggagcgcctg atgcagatgc
     1621 acgagcgctt tgtcgaccgc caagcctcgg ctgctttcaa cgctgccatg gtccgcgcac
     1681 aacggcggat aaaaccggtt gcccgacggg cgcttaatgt tcagaccaat agcacctacg
     1741 cccgactgga agacatcgac cgagagatca gcccgatctt cactgaagaa ggtttctcgc
     1801 tttcgttcgg taccggcgac tcccatctgg ctggttacat tcgggtgatt tgcgacgtga
     1861 tgcatgacca ggggcacacc aggcaataca agatggacct gccgctggac gccaccggca
     1921 ttgggggtaa gacgaacaag accggcgtcc acgctcatgg atccaccaac agttacgccc
     1981 gccgctatct cacgatgaac atcttcaatg tcgtcatggc gaacgaggac accgacggca
     2041 acgcggagcc gcccgaagag cccgtcatca cctcccgcca ggcagcacag ctcgaggcgc
     2101 tgttgaaaaa atgcagccca acgatggctg gcctgttcat tgaaaagtat ggctgcgcgt
     2161 cgaacgtcta caaatccgag ttcgacgagg tactggcgaa gctcaccagg tcagccaatc
     2221 gcactcagca ggagcccgtt gatgcaaatc atcactgagg atcctctaga agaaggagaa
     2281 tataccatga gtggcggttc gcgaattcag ctgctcagcc cgcggctggc caaccagatt
     2341 gccgccggcg aggttgtcga gcggccggcg tctgtggcca aggaactgct gaaaaacagc
     2401 ctggactccg gtgcccggcg catcgatatc gaagtcgagc agggcggcgt caaactgctg
     2461 aaggtgcggg atgatggcag cggcatttct gccgacgacc tgccgctggc cctggctcga
     2521 cacgccacca gcaagatccg cgagctggaa gacctagaag gggtattgag cctgggcttc
     2581 cgtggcgagg ccttggcctc gatcagttcg gtggcgcgcc tgaccctgac ctcgcgtacc
     2641 gccagtgcca gtgaggcctg gcaagtagaa accgaaggcc gcgacatgac gccgcgggtg
     2701 cagccggcgg cgcacccggt cggtacctcg gtggaagtgc gcgatttgtt cttcaatacc
     2761 ccggccaggc gcaagttcct caaagccgag aaaaccgaat tcgatcactt gcaggaagtg
     2821 atccggcggc tggcgctggc ccgtttcgat gtcggcttcc acctgcgcca caatggcaag
     2881 agcatcctca gcctgcacga ggcccacgac gaaatcgccc gtgcacggcg ggtgggtgcc
     2941 atctgcggcc cgggcttcat ggagcaggca ctgccgatcg acgtcgagcg caacggtctg
     3001 cgcctttggg gctgggtcgg cctgccgacg ttctcgcgca gccaggcaga cctgcagtac
     3061 ttcttcgtca atggtcgggc agtgcgcgac aaactggtcg cccatgccgt gcgtcaggcc
     3121 taccgggacg tgctgttcaa cggccgccac cccacgttcg tgctgttcct ggaactggag
     3181 cccaacggcg tcgatgtgaa cgtgcaccca accaagcatg aagtacggtt ccgcgaaggg
     3241 cgctcggtgc atgacttcct gtatggcacc ttgcaccgtg ccttggccga tgtacgtccg
     3301 gaagaccagc tggccgcacc tgctgccgta cctgagctgg ttcgccccac cggccagcag
     3361 gccggtgagt tcgggcccca gggtgaaatg cgcctggcct cgccggtgct tgaacagcct
     3421 cgagccccgc aacagtcgtt ttccaatggt ggcagcggcg ccggttacca gtatcaatac
     3481 accccacgtc cctcgcagcc gctgccggcc gcagaggcac aggcggtgta tcgcgagttc
     3541 tataagccgc tggaaacagg tgctgcgcct gcgacggcac tgcccgaaag ccagggcgac
     3601 attcccccgc taggctatgc cctggcgcag ctcaagggta tctacatcct ggccgagaat
     3661 gctgttggcc tggtgctggt ggacatgcac gccgcccacg agcggataat gtacgagcgc
     3721 ctcaaggtgg ccatggccag cgagggcctg agcggccagc cgctgctggt gccggagacc
     3781 ctggccctca gccagcgtga agccgactgt gccgaagagc atgcacagtg gtttcagcgc
     3841 ctgggcttcg aattgcagcg cctgggcccc gagaccctgg cgattcgcca gatcccggcc
     3901 ttgctcaagc aggctgaagc caaccgcctg gtgcaggacg tgcttgccga ccttatggag
     3961 tacggcacca gtgatcgcat tcaggcgcac ctcaatgagc tgctcggcac catggcttgc
     4021 cacggtgccg tgcgcgccaa ccggcgtctg gcaattcccg agatgaacgc cctgctgcgc
     4081 gacatggaaa acaccgagcg cagcggccag tgcaaccatg gtcgtcctac ctggacccag
     4141 atggggctgg acgatctgga caaacttttc ctgcgcggtc gatgaaagct tgcggccgcg
     4201 tcgtgactgg gaaaaccctg gcgactagtc ttggactcct gttgatagat ccagtaatga
     4261 cctcagaact ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg
     4321 gtgagaatcc aggggtcccc aataattacg atttaaattt gtgtctcaaa atctctgatg
     4381 ttacattgca caagataaaa atatatcatc atgaacaata aaactgtctg cttacataaa
     4441 cagtaataca aggggtgtta tgagccatat tcagcgtgaa acgagctgta gccgtccgcg
     4501 tctgaacagc aacatggatg cggatctgta tggctataaa tgggcgcgtg ataacgtggg
     4561 tcagagcggc gcgaccattt atcgtctgta tggcaaaccg gatgcgccgg aactgtttct
     4621 gaaacatggc aaaggcagcg tggcgaacga tgtgaccgat gaaatggtgc gtctgaactg
     4681 gctgaccgaa tttatgccgc tgccgaccat taaacatttt attcgcaccc cggatgatgc
     4741 gtggctgctg accaccgcga ttccgggcaa aaccgcgttt caggtgctgg aagaatatcc
     4801 ggatagcggc gaaaacattg tggatgcgct ggccgtgttt ctgcgtcgtc tgcatagcat
     4861 tccggtgtgc aactgcccgt ttaacagcga tcgtgtgttt cgtctggccc aggcgcagag
     4921 ccgtatgaac aacggcctgg tggatgcgag cgattttgat gatgaacgta acggctggcc
     4981 ggtggaacag gtgtggaaag aaatgcataa actgctgccg tttagcccgg atagcgtggt
     5041 gacccacggc gattttagcc tggataacct gattttcgat gaaggcaaac tgattggctg
     5101 cattgatgtg ggccgtgtgg gcattgcgga tcgttatcag gatctggcca ttctgtggaa
     5161 ctgcctgggc gaatttagcc cgagcctgca aaaacgtctg tttcagaaat atggcattga
     5221 taatccggat atgaacaaac tgcaatttca tctgatgctg gatgaatttt tctaataatt
     5281 aattggaccg cggtccgcgc gttgtccttt tccgctgcat aaccctgctt cggggtcatt
     5341 atagcgattt tttcggtata tccatccttt ttcgcacgat atacaggatt ttgccaaagg
     5401 gttcgtgtag actttccttg gtgtatccaa cggcgtcagc cgggcaggat aggtgaagta
     5461 ggcccacccg cgagcgggtg ttccttcttc actgtccctt attcgcacct ggcggtgctc
     5521 aacgggaatc ctgctctgcg aggctggccg taggccggcc ctaccggcgc ggcagcgtta
     5581 cccgtgtcgg cggctccaac ggctcgccat cgtccagaaa acacggctca tcgggcatcg
     5641 gcaggcgctg ctgcccgcgc cgttcccatt cctccgtttc ggtcaaggct ggcaggtctg
     5701 gttccatgcc cggaatgccg ggctggctgg gcggctcctc gccggggccg gtcggtagtt
     5761 gctgctcgcc cggatacagg gtcgggatgc ggcgcaggtc gccatgcccc aacagcgatt
     5821 cgtcctggtc gtcgtgatca accaccacgg cggcactgaa caccgacagg cgcaactggt
     5881 cgcggggctg gccccacgcc acgcggtcat tgaccacgta ggccgacacg gtgccggggc
     5941 cgttgagctt cacgacggag atccagcgct cggccaccaa gtccttgact gcgtattgga
     6001 ccgtccgcaa agaacgtccg atgagcttgg aaagtgtctt ctggctgacc accacggcgt
     6061 tctggtggcc catctgcgcc acgaggtgat gcagcagcat tgccgccgtg ggtttcctcg
     6121 caataagccc ggcccacgcc tcatgcgctt tgcgttccgt ttgcacccag tgaccgggct
     6181 tgttcttggc ttgaatgccg atttctctgg actgcgtggc catgcttatc tccatgcggt
     6241 aggggtgccg cacggttgcg gcaccatgcg caatcagctg caacttttcg gcagcgcgac
     6301 aacaattatg cgttgcgtaa aagtggcagt caattacaga ttttctttaa cctacgcaat
     6361 gagctattgc ggggggtgcc gcaatgagct gttgcgtacc cccctttttt aagttgttga
     6421 tttttaagtc tttcgcattt cgccctatat ctagttcttt ggtgcccaaa gaagggcacc
     6481 cctgcggggt tcccccacgc cttcggcgcg gctccccctc cggcaaaaag tggcccctcc
     6541 ggggcttgtt gatcgactgc gcggccttcg gccttgccca aggtggcgct gcccccttgg
     6601 aacccccgca ctcgccgccg tgaggctcgg ggggcaggcg ggcgggcttc gcccttcgac
     6661 tgcccccact cgcataggct tgggtcgttc caggcgcgtc aaggccaagc cgctgcgcgg
     6721 tcgctgcgcg agccttgacc cgccttccac ttggtgtcca accggcaagc gaagcgcgca
     6781 ggccgcaggc cggaggcttt tccccagaga aaattaaaaa aattgatggg gcaaggccgc
     6841 aggccgcgca gttggagccg gtgggtatgt ggtcgaaggc tgggtagccg gtgggcaatc
     6901 cctgtggtca agctcgtggg caggcgcagc ctgtccatca gcttgtccag cagggttgtc
     6961 cacgggccga gcgaagcgag ccagccggtg gccgctcgcg gccatcgtcc acatatccac
     7021 gggctggcaa gggagcgcag cgaccgcgca gggcgaagcc cggagagcaa gcccgtaggg
     7081 ggggcgcgcc cagctgtcta gggcggcgga tttgtcctac tcaggagagc gttcaccgac
     7141 aaacaacaga taaaacgaaa ggcccagtct ttcgactgag cctttcgttt tatttgatgc
     7201 ct
//
