LOCUS       sequence-399780-        6253 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sucrose counter-selection allelic exchange vector for Pseudomonas 
            syringae.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6253)
  TITLE     Kvitko lab Gateway compatible allelic exchange plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6253)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pT18mobsacB
FEATURES             Location/Qualifiers
     source          1..6253
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..446
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(447..533,534..1868)
                     /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
                     ITRHDMLQIPEQQKNEKYQVSEFDSSTIKNISSAKGLDVWDSWPLQNADGTVANYHGYH
                     IVFALAGDPKNADDTSIYMFYQKVGETSIDSWKNAGRVFKDSDKFDANDSILKDQTQEW
                     SGSATFTSDGKIRLFYTDFSGKHYGKQTLTTAQVNVSASDSSLNINGVEDYKSIFDGDG
                     KTYQNVQQFIDEGNYSSGDNHTLRDPHYVEDKGHKYLVFEANTGTEDGYQGEESLFNKA
                     YYGKSTSFFRQESQKLLQSDKKRTAELANGALGMIELNDDYTLKKVMKPLIASNTVTDE
                     IERANVFKMNGKWYLFTDSRGSKMTIDGITSNDIYMLGYVSNSLTGPYKPLNKTGLVLK
                     MDLDPNDVTFTYSHFAVPQAKGNNVVITSYMTNRGFYADKQSTFAPSFLLNIKGKKTSV
                     VKDSILEQGQLTVNK"
     oriT            2222..2331
                     /note="incP origin of transfer"
     promoter        complement(2566..2656)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      3016..3604
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3505..3524
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3758..3775
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3892..3913
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3928..3933,3934..3951,3952..3958)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3966..3982
                     /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     3971..3993
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3990..4006
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3990..4006
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4007..4237
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNRLA
                     AHPPFASWRNSEEARTDRPSQQLRSLNGEWR"
     misc_feature    4015..4071
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(4075..4092)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4075..4091)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4084..4106)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4624..4642
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(4669..4674,4675..4691,4692..4697)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             4745..5935
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(5097..5116)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6078..6097
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
ORIGIN
        1 cacatatacc tgccgttcac tattatttag tgaaatgaga tattatgata ttttctgaat
       61 tgtgattaaa aaggcaactt tatgcccatg caacagaaac tataaaaaat acagagaatg
      121 aaaagaaaca gatagatttt ttagttcttt aggcccgtag tctgcaaatc cttttatgat
      181 tttctatcaa acaaaagagg aaaatagacc agttgcaatc caaacgagag tctaatagaa
      241 tgaggtcgaa aagtaaatcg cgcgggtttg ttactgataa agcaggcaag acctaaaatg
      301 tgtaaagggc aaagtgtata ctttggcgtc accccttaca tattttaggt ctttttttat
      361 tgtgcgtaac taacttgcca tcttcaaaca ggagggctgg aagaagcaga ccgctaacac
      421 agtacataaa aaaggagaca tgaacgatga acatcaaaaa gtttgcaaaa caagcaacag
      481 tattaacctt tactaccgca ctgctggcag gaggcgcaac tcaagcgttt gcgaaagaaa
      541 cgaaccaaaa gccatataag gaaacatacg gcatttccca tattacacgc catgatatgc
      601 tgcaaatccc tgaacagcaa aaaaatgaaa aatatcaagt ttctgaattt gattcgtcca
      661 caattaaaaa tatctcttct gcaaaaggcc tggacgtttg ggacagctgg ccattacaaa
      721 acgctgacgg cactgtcgca aactatcacg gctaccacat cgtctttgca ttagccggag
      781 atcctaaaaa tgcggatgac acatcgattt acatgttcta tcaaaaagtc ggcgaaactt
      841 ctattgacag ctggaaaaac gctggccgcg tctttaaaga cagcgacaaa ttcgatgcaa
      901 atgattctat cctaaaagac caaacacaag aatggtcagg ttcagccaca tttacatctg
      961 acggaaaaat ccgtttattc tacactgatt tctccggtaa acattacggc aaacaaacac
     1021 tgacaactgc acaagttaac gtatcagcat cagacagctc tttgaacatc aacggtgtag
     1081 aggattataa atcaatcttt gacggtgacg gaaaaacgta tcaaaatgta cagcagttca
     1141 tcgatgaagg caactacagc tcaggcgaca accatacgct gagagatcct cactacgtag
     1201 aagataaagg ccacaaatac ttagtatttg aagcaaacac tggaactgaa gatggctacc
     1261 aaggcgaaga atctttattt aacaaagcat actatggcaa aagcacatca ttcttccgtc
     1321 aagaaagtca aaaacttctg caaagcgata aaaaacgcac ggctgagtta gcaaacggcg
     1381 ctctcggtat gattgagcta aacgatgatt acacactgaa aaaagtgatg aaaccgctga
     1441 ttgcatctaa cacagtaaca gatgaaattg aacgcgcgaa cgtctttaaa atgaacggca
     1501 aatggtacct gttcactgac tcccgcggat caaaaatgac gattgacggc attacgtcta
     1561 acgatattta catgcttggt tatgtttcta attctttaac tggcccatac aagccgctga
     1621 acaaaactgg ccttgtgtta aaaatggatc ttgatcctaa cgatgtaacc tttacttact
     1681 cacacttcgc tgtacctcaa gcgaaaggaa acaatgtcgt gattacaagc tatatgacaa
     1741 acagaggatt ctacgcagac aaacaatcaa cgtttgcgcc gagcttcctg ctgaacatca
     1801 aaggcaagaa aacatctgtt gtcaaagaca gcatccttga acaaggacaa ttaacagtta
     1861 acaaataaaa acgcaaaaga aaatgccgat gggtaccgag cgaaatgacc gaccaagcga
     1921 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     1981 tcggaatcgt tttccgggac gccctcgcgg acgtgctcat agtccacgac gcccgtgatt
     2041 ttgtagccct ggccgacggc cagcaggtag gccgacaggc tcatgccggc cgccgccgcc
     2101 ttttcctcaa tcgctcttcg ttcgtctgga aggcagtaca ccttgatagg tgggctgccc
     2161 ttcctggttg gcttggtttc atcagccatc cgcttgccct catctgttac gccggcggta
     2221 gccggccagc ctcgcagagc aggattcccg ttgagcaccg ccaggtgcga ataagggaca
     2281 gtgaagaagg aacacccgct cgcgggtggg cctacttcac ctatcctgcc cggctgacgc
     2341 cgttggatac accaaggaaa gtctacacga accctttggc aaaatcctgt atatcgtgcg
     2401 aaaaaggatg gatataccga aaaaatcgct ataatgaccc cgaagcaggg ttatgcagcg
     2461 gaaaagcgct gcttccctgc tgttttgtgg aatatctacc gactggaaac aggcaaatgc
     2521 aggaaattac tgaactgagg ggacaggcga gagacgatgc caaagagctc ctgaaaatct
     2581 cgataactca aaaaatacgc ccggtagtga tcttatttca ttatggtgaa agttggaacc
     2641 tcttacgtgc cgatcaacgt ctcattttcg ccaaaagttg gcccagggct tcccggtatc
     2701 aacagggaca ccaggattta tttattctgc gaagtgatct tccgtcacag gtatttattc
     2761 ggcgcaaagt gcgtcgggtg atgctgccaa cttactgatt tagtgtatga tggtgttttt
     2821 gaggtgctcc agtggcttct gtttctatca gggctggatg atcctccagc gcggggatct
     2881 catgctggag ttcttcgccc accccaaaag gatctaggtg aagatccttt ttgataatct
     2941 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     3001 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     3061 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     3121 gaaggtaact ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta
     3181 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     3241 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     3301 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3361 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3421 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3481 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3541 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3601 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3661 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3721 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3781 ggaagagcgc ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag
     3841 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
     3901 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
     3961 tggaattgtg agcggataac aatttcacac aggaaacagc tatgacatga ttacgaattc
     4021 gagctcggta cccggggatc ctctagagtc gacctgcagg catgcaagct tggcactggc
     4081 cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc
     4141 agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc
     4201 ccaacagttg cgcagcctga atggcgaatg gcgataagct agcttcacgc tgccgcaagc
     4261 actcagggcg caagggctgc taaaggaagc ggaacacgta gaaagccagt ccgcagaaac
     4321 ggtgctgacc ccggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg
     4381 caaagagaaa gcaggtagct tgcagtgggc ttacatggcg atagctagac tgggcggttt
     4441 tatggacagc aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc
     4501 cctgcaaagt aaactggatg gctttcttgc cgccaaggat ctgatggcgc aggggatcaa
     4561 gatctccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     4621 aaaaataggc gtatcacgag gccctttcgt cttcaagaat tctcatgttt gacagcttat
     4681 catcgataag ctttaatgcg gtagtttatc acagttaaat tgctaacgca gtcaggcacc
     4741 gtgtatgaaa tctaacaatg cgctcatcgt catcctcggc accgtcaccc tggatgctgt
     4801 aggcataggc ttggttatgc cggtactgcc gggcctcttg cgggatatcg tccattccga
     4861 cagcatcgcc agtcactatg gcgtgctgct agcgctatat gcgttgatgc aatttctatg
     4921 cgcacccgtt ctcggagcac tgtccgaccg ctttggccgc cgcccagtcc tgctcgcttc
     4981 gctacttgga gccactatcg actacgcgat catggcgacc acacccgtcc tgtggatcct
     5041 ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg ctggcgccta
     5101 tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca tgagcgcttg
     5161 tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg ccatctcctt
     5221 gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac tgggctgctt
     5281 cctaatgcag gagtcgcata agggagagcg tcgaccgatg cccttgagag ccttcaaccc
     5341 agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta tgactgtctt
     5401 ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt tcggcgagga
     5461 ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg gaatcttgca
     5521 cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg agaagcaggc
     5581 cattatcgcc ggcatggcgg ccgacgcgct gggctacgtc ttgctggcgt tcgcgacgcg
     5641 aggctggatg gccttcccca ttatgattct tctcgcttcc ggcggcatcg ggatgcccgc
     5701 gttgcaggcc atgctgtcca ggcaggtaga tgacgaccat cagggacagc ttcaaggatc
     5761 gctcgcggct cttaccagcc taacttcgat cattggaccg ctgatcgtca cggcgattta
     5821 tgccgcctcg gcgagcacat ggaacgggtt ggcatggatt gtaggcgccg ccctatacct
     5881 tgtctgcctc cccgcgttgc gtcgcggtgc atggagccgg gccacctcga cctgaatgga
     5941 agccggcggc acctcgctaa cggattcacc actccatggc gatgcctgct tgccgaatat
     6001 catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga
     6061 ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg
     6121 ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt
     6181 ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgctag aggatcgatc
     6241 ctttttaacc cat
//
