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"
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 tcagcctgcc gccttgggcc
     5581 gggtgatgtc gtacttgccc gccgcgaact cggttaccgt ccagcccagc gcgaccagct
     5641 ccggcaacgc ctcgcgcacc cgctggcggc gcttgcgcat ggtcgaacca ctggcctctg
     5701 acggccagac atagccgcac aaggtatcta tggaagcctt gccggttttg ccggggtcga
     5761 tccagccaca cagccgctgg tgcagcaggc gggcggtttc gctgtccagc gcccgcacct
     5821 cgtccatgct gatgcgcaca tgctggccgc cacccatgac ggcctgcgcg atcaaggggt
     5881 tcagggccac gtacaggcgc ccgtccgcct cgtcgctggc gtactccgac agcagccgaa
     5941 acccctgccg cttgcggcca ttctgggcga tgatggatac cttccaaagg cgctcgatgc
     6001 agtcctgtat gtgcttgagc gccccaccac tatcgacctc tgccccgatt tcctttgcca
     6061 gcgcccgata gctacctttg accacatggc attcagcggt gacggcctcc cacttgggtt
     6121 ccaggaacag ccggagctgc cgtccgcctt cggtcttggg ttccgggcca agcactaggc
     6181 cattaggccc agccatggcc accagccctt gcaggatgcg cagatcatca gcgcccagcg
     6241 gctccgggcc gctgaactcg atccgcttgc cgtcgccgta gtcatacgtc acgtccagct
     6301 tgctgcgctt gcgctcgccc cgcttgaggg cacggaacag gccgggggcc agacagtgcg
     6361 ccgggtcgtg ccggacgtgg ctgaggctgt gcttgttctt aggcttcacc acggggcacc
     6421 cccttgctct tgcgctgcct ctccagcacg gcgggcttga gcaccccgcc gtcatgccgc
     6481 ctgaaccacc gatcagcgaa cggtgcgcca tagttggcct tgctcacacc gaagcggacg
     6541 aagaaccggc gctggtcgtc gtccacaccc cattcctcgg cctcggcgct ggtcatgctc
     6601 gacaggtagg actgccagcg gatgttatcg accagtaccg agctgccccg gctggcctgc
     6661 tgctggtcgc ctgcgcccat catggccgcg cccttgctgg catggtgcag gaacacgata
     6721 gagcacccgg tatcggcggc gatggcctcc atgcgaccga tgacctgggc catggggccg
     6781 ctggcgtttt cttcctcgat gtggaaccgg cgcagcgtgt ccagcaccat caggcggcgg
     6841 ccctcggcgg cgcgcttgag gccgtcgaac cactccgggg ccatgatgtt gggcaggctg
     6901 ccgatcagcg gctggatcag caggccgtca gccacggctt gccgttcctc ggcgctgagg
     6961 tgcgccccaa gggcgtgcag gcggtgatga atggcggtgg gcgggtcttc ggcgggcagg
     7021 tagatcaccg ggccggtggg cagttcgccc acctccagca gatccggccc gcctgcaatc
     7081 tgtgcggcca gttgcagggc cagcatggat ttaccggcac caccgggcga caccagcgcc
     7141 ccgaccgtac cggccaccat gttgggcaaa acgtagtcca gcggtggcgg cgctgctgcg
     7201 aacgcctcca gaatattgat aggcttatgg gtagccattg attgcctcct ttgcaggcag
     7261 ttggtggtta ggcgctggcg gggtcactac ccccgccctg cgccgctctg agttcttcca
     7321 ggcactcgcg cagcgcctcg tattcgtcgt cggtcagcca gaacttgcgc tgacgcatcc
     7381 ctttggcctt catgcgctcg gcatatcgcg cttggcgtac agcgtcaggg ctggccagca
     7441 ggtcgccggt ctgcttgtcc ttttggtctt tcatatcagt caccgagaaa cttgccgggg
     7501 ccgaaaggct tgtcttcgcg gaacaaggac aaggtgcagc cgtcaaggtt aaggctggcc
     7561 atatcagcga ctgaaaagcg gccagcctcg gccttgtttg acgtataacc aaagccaccg
     7621 ggcaaccaat agcccttgtc acttttgatc aggtagaccg accctgaagc gcttttttcg
     7681 tattccataa aacccccttc tgtgcgtgag tactcatagt ataacaggcg tgagtaccaa
     7741 cgcaagcact acatgctgaa atctggcccg cccctgtcca tgcctcgctg gcggggtgcc
     7801 ggtgcccgtg ccagctcggc ccgcgcaagc tggacgctgg gcagacccat gaccttgctg
     7861 acggtgcgct cgatgtaatc cgcttcgtgg ccgggcttgc gctctgccag cgctgggctg
     7921 gcctcggcca tggccttgcc gatttcctcg gcactgcggc cccggctggc cagcttctgc
     7981 gcggcgataa agtcgcactt gctgaggtca tcaccgaagc gcttgaccag cccggccatc
     8041 tcgctgcggt actcgtccag cgccgtgcgc cggtggcggc taagctgccg ctcgggcagt
     8101 tcgaggctgg ccagcctgcg ggccttctcc tgctgccgct gggcctgctc gatctgctgg
     8161 ccagcctgct gcaccagcgc cgggccagcg gtggcggtct tgcccttgga ttcacgcagc
     8221 agcacccacg gctgataacc ggcgcgggtg gtgtgcttgt ccttgcggtt ggtgaagccc
     8281 gccaagcggc catagtggcg gctgtcggcg ctggccgggt cggcgtcgta ctcgctggcc
     8341 agcgtccggg caatctgccc ccgaagttca ccgcctgcgg cgtcggccac cttgacccat
     8401 gcctgatagt tcttcgggct ggtttccact accagggcag gctcccggcc ctcggctttc
     8461 atgtcatcca ggtcaaactc gctgaggtcg tccaccagca ccagaccatg ccgctcctgc
     8521 tcggcgggcc tgatatacac gtcattgccc tgggcattca tccgcttgag ccatggcgtg
     8581 ttctggagca cttcggcggc tgaccattcc cggttcatca tctggccggt ggtggcgtcc
     8641 ctgacgccga tatcgaagcg ctcacagccc atggccttga gctgtcggcc tatggcctgc
     8701 aaagtcctgt cgttcttcat cgggccacca agcgattccc acacattata cgagccggaa
     8761 gcataaagtg taaagcctag atccgaagga tgagccgggc tgaatgatcg accgagacag
     8821 gccctgcggg gctgcacacg cgcccccacc cttcgggtag ggggaaaggc cgctaaagcg
     8881 gctaaaagcg ctccagcgta tttctgcggg gtttggtgtg gggtttagcg ggctttgccc
     8941 gcctttcccc ctgccgcgca gcggtggggc ggtgtgtagc ctagcgcagc gaatagacca
     9001 gctatccggc ctctggccgg gcatattggg caagggcagc agcgccccac aagggcgctg
     9061 ataaccgcgc ctagtggatt attcttagat aatcatggat ggatttttcc aacaccccgc
     9121 cagcccccgc ccctgctggg tttgcaggtt tgggggcgtg acagttattg caggggttcg
     9181 tgacagttat tgcagggggg cgtgacagtt attgcagggg ttcgtgacag ttagggcgcg
     9241 cccagctgtc tagggcggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca
     9301 gataaaacga aaggcccagt ctttcgactg agcctttcgt tttatttgat gcct
//
