LOCUS       sequence-243571-        4451 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  constitutive expression of I-SceI for genome engineering in 
            Pseudomonas.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4451)
  AUTHORS   Wirth NT, Kozaeva E, Nikel PI
  TITLE     Accelerated genome engineering of Pseudomonas putida by 
            I-SceI-mediated recombination and CRISPR-Cas9 counterselection.
  JOURNAL   Microb Biotechnol. 2019 Mar 12. doi: 10.1111/1751-7915.13396.
  PUBMED    30861315
REFERENCE   2  (bases 1 to 4451)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Microb 
            Biotechnol. 2019 Mar 12. doi: 10.1111/1751-7915.13396."
COMMENT     Sequence Label: pSEVA6213S
FEATURES             Location/Qualifiers
     source          1..4451
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    809..865
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     terminator      908..1002
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     promoter        join(1033..1038,1039..1055,1056..1061)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             1250..1783
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     oriT            1994..2102
                     /note="incP origin of transfer"
     CDS             complement(2163..3311)
                     /codon_start=1
                     /product="trans-acting replication protein that binds to 
                     and activates oriV"
                     /label=trfA
                     /translation="MNRTFDRKAYRQELIDAGFSAEDAETIVSRTVMRAPRETFQSVGS
                     IVQQATAKIERDSVQLAPPALPAPSAAVERSRRLEQEAAGLAKSMTIDTRGTMTTKKRK
                     TAGEDLAKQVSEAKQAALLKHTKQQIKEMQLSLFDIAPWPDTMRAMPNDTARSALFTTR
                     NKKIPREALQNKVIFHVNKDVKITYTGVELRADDDELVWQQVLEYAKRTPIGEPITFTF
                     YELCQDLGWSINGRYYTKAEECLSRLQATAMGFTSDRVGHLESVSLLHRFRVLDRGKKT
                     SRCQVLIDEEIVVLFAGDHYTKFIWEKYRKLSPTARRMFDYFSSHREPYPLKLETFRLM
                     CGSDSTRVKKWREQVGEACEELRGSGLVEHAWVNDDLVHCKR"
     rep_origin      3704..4235
                     /label=oriV
                     /note="incP origin of replication"
     terminator      complement(4362..4448)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
ORIGIN
        1 cctttaatta atgtgacaat taatcatcgg catagtatat cggcatagta taatacgaca
       61 aggtgaggaa ctaaacccct aggggagtca tgaacatatg aaaaacatca aaaaaaacca
      121 ggtaatgaac ctgggtccga actctaaact gctgaaagaa tacaaatccc agctgatcga
      181 actgaacatc gaacagttcg aagcaggtat cggtctgatc ctgggtgatg cttacatccg
      241 ttctcgtgat gaaggtaaaa cctactgtat gcagttcgag tggaaaaaca aagcatacat
      301 ggaccacgta tgtctgctgt acgatcagtg ggtactgtcc ccgccgcaca aaaaagaacg
      361 tgttaaccac ctgggtaacc tggtaatcac ctggggcgcc cagactttca aacaccaggc
      421 tttcaacaaa ctggctaacc tgttcatcgt taacaacaaa aaaaccatcc cgaacaacct
      481 ggttgaaaac tacctgaccc cgatgtctct ggcatactgg ttcatggatg atggtggtaa
      541 atgggattac aacaaaaact ctaccaacaa atcgatcgta ctgaacaccc agtctttcac
      601 tttcgaagaa gtagaatacc tggttaaggg tctgcgtaac aaattccaac tgaactgtta
      661 cgtaaaaatc aacaaaaaca aaccgatcat ctacatcgat tctatgtctt acctgatctt
      721 ctacaacctg atcaaaccgt acctgatccc gcagatgatg tacaaactgc cgaacactat
      781 ctcctccgaa actttcctga aataataaga attcgagctc ggtacccggg gatcctctag
      841 agtcgacctg caggcatgca agcttgcggc cgcgtcgtga ctgggaaaac cctggcgact
      901 agtcttggac tcctgttgat agatccagta atgacctcag aactccatct ggatttgttc
      961 agaacgctcg gttgccgccg ggcgtttttt attggtgaga atccaggggt ccccaataat
     1021 tacgatttaa atttgacata agcctgttcg gttcgtaaac tgtaatgcaa gtagcgtatg
     1081 cgctcacgca actggtccag aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg
     1141 gttttcatgg cttgttatga ctgttttttt gtacagccta tgcctcgggc atccaagcag
     1201 caagcgcgtt acgccgtggg tcgatgtttg atgttatgga gcagcaacga tgttacgcag
     1261 cagcaacgat gttacgcagc agggcagtcg ccctaaaaca aagttaggtg gctcaagtat
     1321 gggcatcatt cgcacatgta ggctcggccc tgaccaagtc aaatccatgc gggctgctct
     1381 tgatcttttc ggtcgtgagt tcggagacgt agccacctac tcccaacatc agccggactc
     1441 cgattacctc gggaacttgc tccgtagtaa gacattcatc gcgcttgctg ccttcgacca
     1501 agaagcggtt gttggcgctc tcgcggctta cgttctgccc aagtttgagc agccgcgtag
     1561 tgagatctat atctatgatc tcgcagtctc cggagagcac cggaggcagg gcattgccac
     1621 cgcgctcatc aatctcctca agcatgaggc caacgcgctt ggtgcttatg tgatctacgt
     1681 gcaagcagat tacggtgacg atcccgcagt ggctctctat acaaagttgg gcatacggga
     1741 agaagtgatg cactttgata tcgacccaag taccgccacc taacaattcg ttcaagccga
     1801 gatcggcttc ccggccgcgg agttgttcgg taaattggac aacggtccgc gcgttgtcct
     1861 tttccgctgc ataaccctgc ttcggggtca ttatagcgat tttttcggta tatccatcct
     1921 ttttcgcacg atatacagga ttttgccaaa gggttcgtgt agactttcct tggtgtatcc
     1981 aacggcgtca gccgggcagg ataggtgaag taggcccacc cgcgagcggg tgttccttct
     2041 tcactgtccc ttattcgcac ctggcggtgc tcaacgggaa tcctgctctg cgaggctggc
     2101 cgtaggccgg ccgcgatgca ggtggctgct gaacccccag ccggaactga ccccacaagg
     2161 ccctagcgtt tgcaatgcac caggtcatca ttgacccagg cgtgttccac caggccgctg
     2221 cctcgcaact cttcgcaggc ttcgccgacc tgctcgcgcc acttcttcac gcgggtggaa
     2281 tccgatccgc acatgaggcg gaaggtttcc agcttgagcg ggtacggctc ccggtgcgag
     2341 ctgaaatagt cgaacatccg tcgggccgtc ggcgacagct tgcggtactt ctcccatatg
     2401 aatttcgtgt agtggtcgcc agcaaacagc acgacgattt cctcgtcgat caggacctgg
     2461 caacgggacg ttttcttgcc acggtccagg acgcggaagc ggtgcagcag cgacaccgat
     2521 tccaggtgcc caacgcggtc ggacgtgaag cccatcgccg tcgcctgtag gcgcgacagg
     2581 cattcctcgg ccttcgtgta ataccggcca ttgatcgacc agcccaggtc ctggcaaagc
     2641 tcgtagaacg tgaaggtgat cggctcgccg ataggggtgc gcttcgcgta ctccaacacc
     2701 tgctgccaca ccagttcgtc atcgtcggcc cgcagctcga cgccggtgta ggtgatcttc
     2761 acgtccttgt tgacgtggaa aatgaccttg ttttgcagcg cctcgcgcgg gattttcttg
     2821 ttgcgcgtgg tgaacagggc agagcgggcc gtgtcgtttg gcatcgctcg catcgtgtcc
     2881 ggccacggcg caatatcgaa caaggaaagc tgcatttcct tgatctgctg cttcgtgtgt
     2941 ttcagcaacg cggcctgctt ggcttcgctg acctgttttg ccaggtcctc gccggcggtt
     3001 tttcgcttct tggtcgtcat agttcctcgc gtgtcgatgg tcatcgactt cgccaaacct
     3061 gccgcctcct gttcgagacg acgcgaacgc tccacggcgg ccgatggcgc gggcagggca
     3121 gggggagcca gttgcacgct gtcgcgctcg atcttggccg tagcttgctg gactatcgag
     3181 ccgacggact ggaaggtttc gcggggcgca cgcatgacgg tgcggcttac gatggtttcg
     3241 gcatcctcgg cggaaaaccc cgcgtcgatc agttcttgcc tgtatgcctt ccggtcaaac
     3301 gtccgattca ttcaccctcc ttgcgggatt gccccggaat taattccccg gatcgatccg
     3361 tcgatcttga tcccctgcgc catcagatcc ttggcggcaa gaaagccatc cagtttactt
     3421 tgcagggctt cccaacctta ccagagggcg ccccagctgg caattccggt tcgcttgctg
     3481 tccataaaac cgcccagtct agctatcgcc atgtaagccc actgcaagct acctgctttc
     3541 tctttgcgct tgcgttttcc cttgtccaga tagcccagta gctgacattc atccggggtc
     3601 agcaccgttt ctgcggactg gctttctacg tggctgccat ttttggggtg aggccgttcg
     3661 cggccgaggg gcgcagcccc tggggggatg ggaggcccgc gttagcgggc cgggagggtt
     3721 cgagaagggg gggcaccccc cttcggcgtg cgcggtcacg cgcacagggc gcagccctgg
     3781 ttaaaaacaa ggtttataaa tattggttta aaagcaggtt aaaagacagg ttagcggtgg
     3841 ccgaaaaacg ggcggaaacc cttgcaaatg ctggattttc tgcctgtgga cagcccctca
     3901 aatgtcaata ggtgcgcccc tcatctgtca gcactctgcc cctcaagtgt caaggatcgc
     3961 gcccctcatc tgtcagtagt cgcgcccctc aagtgtcaat accgcagggc acttatcccc
     4021 aggcttgtcc acatcatctg tgggaaactc gcgtaaaatc aggcgttttc gccgatttgc
     4081 gaggctggcc agctccacgt cgccggccga aatcgagcct gcccctcatc tgtcaacgcc
     4141 gcgccgggtg agtcggcccc tcaagtgtca acgtccgccc ctcatctgtc agtgagggcc
     4201 aagttttccg cgaggtatcc acaacgccgg cggccctaca tggctctgct gtagtgagtg
     4261 ggttgcgctc cggcagcggt cctgatcccc cgcagaaaaa aaggatctca agaagatcct
     4321 ttgatctttt ctacggcgcg cccagctgtc tagggcggcg gatttgtcct actcaggaga
     4381 gcgttcaccg acaaacaaca gataaaacga aaggcccagt ctttcgactg agcctttcgt
     4441 tttatttgat g
//
