LOCUS       sequence-502947-        6239 bp    DNA     circular SYN 13-MAR-2026
DEFINITION  Tetracycline resistant derivative of pKEK2201 for allelic exchange .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6239)
  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 6239)
  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: pVAM03
FEATURES             Location/Qualifiers
     source          1..6239
                     /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(2362..2384)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             2574..3764
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     CDS             complement(join(3797..5131,5132..5218))
                     /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(5219..5664)
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     protein_bind    5788..5809
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5824..5829,5830..5847,5848..5854)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5862..5878
                     /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     5867..5889
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5886..5902
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5886..5902
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             5898..6239
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITAAANSSSVPGDPLESTCRHASLGPALAVVLQRRDWENPGV
                     TQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIRQSN
                     HSTRPVAAH"
     misc_feature    5921..5977
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(5987..6004)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5987..6003)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5996..6018)
                     /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 catatgaaat
     2581 ctaacaatgc gctcatcgtc atcctcggca ccgtcaccct ggatgctgta ggcataggct
     2641 tggttatgcc ggtactgccg ggcctcttgc gggatatcgt ccattccgac agcatcgcca
     2701 gtcactatgg cgtgctgcta gcgctatatg cgttgatgca atttctatgc gcacccgttc
     2761 tcggagcact gtccgaccgc tttggccgcc gcccagtcct gctcgcttcg ctacttggag
     2821 ccactatcga ctacgcgatc atggcgacca cacccgtcct gtggatcctc tacgccggac
     2881 gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca
     2941 tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg
     3001 gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat
     3061 tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg
     3121 agtcgcataa gggagagcgt cgaccgatgc ccttgagagc cttcaaccca gtcagctcct
     3181 tccggtgggc gcggggcatg actatcgtcg ccgcacttat gactgtcttc tttatcatgc
     3241 aactcgtagg acaggtgccg gcagcgctct gggtcatttt cggcgaggac cgctttcgct
     3301 ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg aatcttgcac gccctcgctc
     3361 aagccttcgt cactggaccc gccaccaaac gtttcggcga gaagcaggcc attatcgccg
     3421 gcatggcggc cgacgcgctg ggctacgtct tgctggcgtt cgcgacgcga ggctggatgg
     3481 ccttccccat tatgattctt ctcgcttccg gcggcatcgg gatgcccgcg ttgcaggcca
     3541 tgctgtccag gcaggtagat gacgaccatc agggacagct tcaaggatcg ctcgcggctc
     3601 ttaccagcct aacttcgatc attggaccgc tgatcgtcac ggcgatttat gccgcctcgg
     3661 cgagcacatg gaacgggttg gcatggattg taggcgccgc cctatacctt gtctgcctcc
     3721 ccgcgttgcg tcgcggtgca tggagccggg ccacctcgac ctgaatggaa gcccatcggc
     3781 attttctttt gcgtttttat ttgttaactg ttaattgtcc ttgttcaagg atgctgtctt
     3841 tgacaacaga tgttttcttg cctttgatgt tcagcaggaa gcttggcgca aacgttgatt
     3901 gtttgtctgc gtagaatcct ctgtttgtca tatagcttgt aatcacgaca ttgtttcctt
     3961 tcgcttgagg tacagcgaag tgtgagtaag taaaggttac atcgttagga tcaagatcca
     4021 tttttaacac aaggccagtt ttgttcagcg gcttgtatgg gccagttaaa gaattagaaa
     4081 cataaccaag catgtaaata tcgttagacg taatgccgtc aatcgtcatt tttgatccgc
     4141 gggagtcagt gaacaggtac catttgccgt tcattttaaa gacgttcgcg cgttcaattt
     4201 catctgttac tgtgttagat gcaatcagcg gtttcatcac ttttttcagt gtgtaatcat
     4261 cgtttagctc aatcataccg agagcgccgt ttgctaactc agccgtgcgt tttttatcgc
     4321 tttgcagaag tttttgactt tcttgacgga agaatgatgt gcttttgcca tagtatgctt
     4381 tgttaaataa agattcttcg ccttggtagc catcttcagt tccagtgttt gcttcaaata
     4441 ctaagtattt gtggccttta tcttctacgt agtgaggatc tctcagcgta tggttgtcgc
     4501 ctgagctgta gttgccttca tcgatgaact gctgtacatt ttgatacgtt tttccgtcac
     4561 cgtcaaagat tgatttataa tcctctacac cgttgatgtt caaagagctg tctgatgctg
     4621 atacgttaac ttgtgcagtt gtcagtgttt gtttgccgta atgtttaccg gagaaatcag
     4681 tgtagaataa acggattttt ccgtcagatg taaatgtggc tgaacctgac cattcttgtg
     4741 tttggtcttt taggatagaa tcatttgcat cgaatttgtc gctgtcttta aagacgcggc
     4801 cagcgttttt ccagctgtca atagaagttt cgccgacttt ttgatagaac atgtaaatcg
     4861 atgtgtcatc cgcattttta ggatctccgg ctaatgcaaa gacgatgtgg tagccgtgat
     4921 agtttgcgac agtgccgtca gcgttttgta atggccagct gtcccaaacg tccaggcctt
     4981 ttgcagaaga gatattttta attgtggacg aatcgaattc aggaacttga tatttttcat
     5041 ttttttgctg ttcagggatt tgcagcatat catggcgtgt aatatgggaa atgccgtatg
     5101 tttccttata tggcttttgg ttcgtttctt tcgcaaacgc ttgagttgcg cctcctgcca
     5161 gcagtgcggt agtaaaggtt aatactgttg cttgttttgc aaactttttg atgttcatcg
     5221 ttcatgtctc cttttttatg tactgtgtta gcggtctgct tcttccagcc ctcctgtttg
     5281 aagatggcaa gttagttacg cacaataaaa aaagacctaa aatatgtaag gggtgacgcc
     5341 aaagtataca ctttgccctt tacacatttt aggtcttgcc tgctttatca gtaacaaacc
     5401 cgcgcgattt acttttcgac ctcattctat tagactctcg tttggattgc aactggtcta
     5461 ttttcctctt ttgtttgata gaaaatcata aaaggatttg cagactacgg gcctaaagaa
     5521 ctaaaaaatc tatctgtttc ttttcattct ctgtattttt tatagtttct gttgcatggg
     5581 cataaagttg cctttttaat cacaattcag aaaatatcat aatatctcat ttcactaaat
     5641 aatagtgaac ggcaggtata tgtgatgggt taaaaaggat cagcgcccaa tacgcaaacc
     5701 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     5761 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     5821 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     5881 tcacacagga aacagctatg accatgatta ctgcggccgc gaattcgagc tcggtacccg
     5941 gggatcctct agagtcgacc tgcaggcatg caagcttggg cccggcactg gccgtcgttt
     6001 tacaacgtcg tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc
     6061 cccctttcgc cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt
     6121 tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg
     6181 gtatttcaca ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaa
//
