LOCUS       sequence-502955-        5580 bp    DNA     circular SYN 13-MAR-2026
DEFINITION  Gentamicin resistant derivative of pKEK2201 for allelic exchange.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5580)
  AUTHORS   Van Allen ME, Chandrashekarappa DG, Bina XR, Bina JE
  TITLE     New vectors and optimal conditions for allelic exchange in 
            hypervirulent Klebsiella pneumoniae.
  JOURNAL   J Microbiol Methods. 2025 Jan;228:107070. doi: 
            10.1016/j.mimet.2024.107070. Epub 2024 Nov 19.
  PUBMED    39566662
REFERENCE   2  (bases 1 to 5580)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Mar 13, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Microbiol
            Methods."; date: "2025-01"; volume: "228:107070. doi"; pages: " 
            10.1016/j.mimet.2024.107070. Epub 2024 Nov 19"
COMMENT     Sequence Label: pVAM05
FEATURES             Location/Qualifiers
     source          1..5580
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      4..392
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(1010..1381)
                     /codon_start=1
                     /gene="traJ"
                     /product="oriT-recognizing protein"
                     /label=traJ
                     /translation="MADETKPTRKGSPPIKVYCLPDERRAIEEKAAAAGMSLSAYLLAV
                     GQGYKITGVVDYEHVRELARINGDLGRLGGLLKLWLTDDPRTARFGDATILALLAKIEE
                     KQDELGKVMMGVVRPRAEP"
     oriT            1414..1523
                     /note="incP origin of transfer"
     primer_bind     complement(2204..2223)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(2362..2384)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             2574..3107
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     CDS             complement(join(3138..4472,4473..4559))
                     /codon_start=1
                     /gene="Bacillus subtilis sacB"
                     /product="secreted levansucrase that renders bacterial 
                     growth sensitive to sucrose"
                     /label=SacB
                     /note="negative selection marker"
                     /translation="MNIKKFAKQATVLTFTTALLAGGATQAFAKETNQKPYKETYGISH
                     ITRHDMLQIPEQQKNEKYQVPEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     promoter        complement(4560..5005)
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     protein_bind    5129..5150
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5165..5170,5171..5188,5189..5195)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5203..5219
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     5208..5230
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5227..5243
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5227..5243
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5239..5580
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITAAANSSSVPGDPLESTCRHASLGPALAVVLQRRDWENPGV
                     TQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIRQSN
                     HSTRPVAAH"
     misc_feature    5262..5318
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(5328..5345)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5328..5344)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5337..5359)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ccatgtcagc cgttaagtgt tcctgtgtca ctcaaaattg ctttgagagg ctctaagggc
       61 ttctcagtgc gttacatccc tggcttgttg tccacaaccg ttaaacctta aaagctttaa
      121 aagccttata tattcttttt tttcttataa aacttaaaac cttagaggct atttaagttg
      181 ctgatttata ttaattttat tgttcaaaca tgagagctta gtacgtgaaa catgagagct
      241 tagtacgtta gccatgagag cttagtacgt tagccatgag ggtttagttc gttaaacatg
      301 agagcttagt acgttaaaca tgagagctta gtacgtgaaa catgagagct tagtacgtac
      361 tatcaacagg ttgaactgct gatcttcaga tcctctacgc cggacgcatc gtggccggat
      421 ccagccgacc aggctttcca cgcccgcgtg ccgctccatg tcgttcgcgc ggttctcgga
      481 aacgcgctgc cgcgtttcgt gattgtcacg ctcaagcccg tagtcccgtt cgagcgtcgc
      541 gcagaggtca gcgagggcgc ggtaggcccg atacggctca tggatggtgt ttcgggtcgg
      601 gtgaatcttg ttgatggcga tatggatgtg caggttgtcg gtgtcgtgat gcacggcact
      661 gacgcgctga tgctcggcga agccaagccc agcgcagatg cggtcctcaa tcgcgcgcaa
      721 cgtctccgcg tcgggcttct ctcccgcgcg gaagctaacc agcaggtgat aggtcttgtc
      781 ggcctcggaa cgggtgttgc cgtgctgggt cgccatcacc tcggccatga cagcgggcag
      841 ggtgtttgcc tcgcagttcg tgacgcgcac gtgacccagg cgctcggtct tgccttgctc
      901 gtcggtgatg tacttcacca gctccgcgaa gtcgctcttc ttgatggagc gcatggggac
      961 gtgcttggca atcacgcgca ccccccggcc gttttagcgg ctaaaaaagt catggctctg
     1021 ccctcgggcg gaccacgccc atcatgacct tgccaagctc gtcctgcttc tcttcgatct
     1081 tcgccagcag ggcgaggatc gtggcatcac cgaaccgcgc cgtgcgcggg tcgtcggtga
     1141 gccagagttt cagcaggccg cccaggcggc ccaggtcgcc attgatgcgg gccagctcgc
     1201 ggacgtgctc atagtccacg acgcccgtga ttttgtagcc ctggccgacg gccagcaggt
     1261 aggccgacag gctcatgccg gccgccgccg ccttttcctc aatcgctctt cgttcgtctg
     1321 gaaggcagta caccttgata ggtgggctgc ccttcctggt tggcttggtt tcatcagcca
     1381 tccgcttgcc ctcatctgtt acgccggcgg tagccggcca gcctcgcaga gcaggattcc
     1441 cgttgagcac cgccaggtgc gaataaggga cagtgaagaa ggaacacccg ctcgcgggtg
     1501 ggcctacttc acctatcctg cccggctgac gccgttggat acaccaagga aagtctacac
     1561 gaaccctttg gcaaaatcct gtatatcgtg cgaaaaagga tggatatacc gaaaaaatcg
     1621 ctataatgac cccgaagcag ggttatgcag cggaaaagcg ctgcttccct gctgttttgt
     1681 ggaatatcta ccgactggaa acaggcaaat gcaggaaatt actgaactga ggggacaggc
     1741 gagagacgat gccaaagagc tacaccgacg agctggccga gtgggttgaa tcccgcgcgg
     1801 ccaagaagcg ccggcgtgat gaggctgcgg ttgcgttcct ggcggtgagg gcggatgtcg
     1861 aggcggcgtt agcgtccggc tatgcgctcg tcaccatttg ggagcacatg cgggaaacgg
     1921 ggaaggtcaa gttctcctac gagacgttcc gctcgcacgc caggcggcac atcaaggcca
     1981 agcccgccga tgtgcccgca ccgcaggcca aggctgcgga acccgcgccg gcacccaaga
     2041 cgccggagcc acggcggccg aagcaggggg gcaaggctga aaagccggcc cccgctgcgg
     2101 ccccgaccgg cttcaccttc aacccaacac cggacaaaaa ggatcctcta cgccggacgc
     2161 atcgtggccg gcatcaccgg cgccacaggt gcggttgctg gcgcctatat cgccgacatc
     2221 accgatgggg aagatcgggc tcgccacttc gggctcatga gcgcttgttt cggcgtgggt
     2281 atggtggcag gccccgtggc cgggggactg ttgggcgcca tctccttgct gcctcgcgcg
     2341 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     2401 tctgtaagcg gtttgggttg tcactcatcg tatttggttt ataattttaa gctaataacc
     2461 taattataac taattaatag ttttgtatct tgaaaaaata gctataaaac ttatttaaat
     2521 aacgaagatt tttgtgtata aaatatttat aacaaaaaaa ggagactaaa catatgttac
     2581 gcagcagcaa cgatgttacg cagcagggca gtcgccctaa aacaaagtta ggtggctcaa
     2641 gtatgggcat cattcgcaca tgtaggctcg gccctgacca agtcaaatcc atgcgggctg
     2701 ctcttgatct tttcggtcgt gagttcggag acgtagccac ctactcccaa catcagccgg
     2761 actccgatta cctcgggaac ttgctccgta gtaagacatt catcgcgctt gctgccttcg
     2821 accaagaagc ggttgttggc gctctcgcgg cttacgttct gcccaagttt gagcagccgc
     2881 gtagtgagat ctatatctat gatctcgcag tctccggaga gcaccggagg cagggcattg
     2941 ccaccgcgct catcaatctc ctcaagcatg aggccaacgc gcttggtgct tatgtgatct
     3001 acgtgcaagc agattacggt gacgatcccg cagtggctct ctatacaaag ttgggcatac
     3061 gggaagaagt gatgcacttt gatatcgacc caagtaccgc cacctaacaa ttcccatcgg
     3121 cattttcttt tgcgttttta tttgttaact gttaattgtc cttgttcaag gatgctgtct
     3181 ttgacaacag atgttttctt gcctttgatg ttcagcagga agcttggcgc aaacgttgat
     3241 tgtttgtctg cgtagaatcc tctgtttgtc atatagcttg taatcacgac attgtttcct
     3301 ttcgcttgag gtacagcgaa gtgtgagtaa gtaaaggtta catcgttagg atcaagatcc
     3361 atttttaaca caaggccagt tttgttcagc ggcttgtatg ggccagttaa agaattagaa
     3421 acataaccaa gcatgtaaat atcgttagac gtaatgccgt caatcgtcat ttttgatccg
     3481 cgggagtcag tgaacaggta ccatttgccg ttcattttaa agacgttcgc gcgttcaatt
     3541 tcatctgtta ctgtgttaga tgcaatcagc ggtttcatca cttttttcag tgtgtaatca
     3601 tcgtttagct caatcatacc gagagcgccg tttgctaact cagccgtgcg ttttttatcg
     3661 ctttgcagaa gtttttgact ttcttgacgg aagaatgatg tgcttttgcc atagtatgct
     3721 ttgttaaata aagattcttc gccttggtag ccatcttcag ttccagtgtt tgcttcaaat
     3781 actaagtatt tgtggccttt atcttctacg tagtgaggat ctctcagcgt atggttgtcg
     3841 cctgagctgt agttgccttc atcgatgaac tgctgtacat tttgatacgt ttttccgtca
     3901 ccgtcaaaga ttgatttata atcctctaca ccgttgatgt tcaaagagct gtctgatgct
     3961 gatacgttaa cttgtgcagt tgtcagtgtt tgtttgccgt aatgtttacc ggagaaatca
     4021 gtgtagaata aacggatttt tccgtcagat gtaaatgtgg ctgaacctga ccattcttgt
     4081 gtttggtctt ttaggataga atcatttgca tcgaatttgt cgctgtcttt aaagacgcgg
     4141 ccagcgtttt tccagctgtc aatagaagtt tcgccgactt tttgatagaa catgtaaatc
     4201 gatgtgtcat ccgcattttt aggatctccg gctaatgcaa agacgatgtg gtagccgtga
     4261 tagtttgcga cagtgccgtc agcgttttgt aatggccagc tgtcccaaac gtccaggcct
     4321 tttgcagaag agatattttt aattgtggac gaatcgaatt caggaacttg atatttttca
     4381 tttttttgct gttcagggat ttgcagcata tcatggcgtg taatatggga aatgccgtat
     4441 gtttccttat atggcttttg gttcgtttct ttcgcaaacg cttgagttgc gcctcctgcc
     4501 agcagtgcgg tagtaaaggt taatactgtt gcttgttttg caaacttttt gatgttcatc
     4561 gttcatgtct ccttttttat gtactgtgtt agcggtctgc ttcttccagc cctcctgttt
     4621 gaagatggca agttagttac gcacaataaa aaaagaccta aaatatgtaa ggggtgacgc
     4681 caaagtatac actttgccct ttacacattt taggtcttgc ctgctttatc agtaacaaac
     4741 ccgcgcgatt tacttttcga cctcattcta ttagactctc gtttggattg caactggtct
     4801 attttcctct tttgtttgat agaaaatcat aaaaggattt gcagactacg ggcctaaaga
     4861 actaaaaaat ctatctgttt cttttcattc tctgtatttt ttatagtttc tgttgcatgg
     4921 gcataaagtt gcctttttaa tcacaattca gaaaatatca taatatctca tttcactaaa
     4981 taatagtgaa cggcaggtat atgtgatggg ttaaaaagga tcagcgccca atacgcaaac
     5041 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     5101 ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc
     5161 aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat
     5221 ttcacacagg aaacagctat gaccatgatt actgcggccg cgaattcgag ctcggtaccc
     5281 ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg gcccggcact ggccgtcgtt
     5341 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     5401 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     5461 ttgcgcagcc tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc
     5521 ggtatttcac accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa
//
