LOCUS       sequence-495901-        6537 bp    DNA     circular SYN 19-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 6537)
  TITLE     Kvitko lab Gateway compatible allelic exchange plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6537)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 19, 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            "Unpublished"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..6537
                     /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"
     promoter        complement(2850..2940)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      3300..3888
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3789..3808
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4042..4059
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4176..4197
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4212..4217,4218..4235,4236..4242)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4250..4266
                     /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     4255..4277
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4274..4290
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4274..4290
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4291..4521
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MITNSSSVPGDPLESTCRHASLALAVVLQRRDWENPGVTQLNRLA
                     AHPPFASWRNSEEARTDRPSQQLRSLNGEWR"
     misc_feature    4299..4355
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(4359..4376)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4359..4375)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4368..4390)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     4908..4926
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(4953..4958,4959..4975,4976..4981)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             5029..6219
                     /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(5381..5400)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6362..6381
                     /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 gctcctgaaa atctcgataa ctcaaaaaat
     2881 acgcccggta gtgatcttat ttcattatgg tgaaagttgg aacctcttac gtgccgatca
     2941 acgtctcatt ttcgccaaaa gttggcccag ggcttcccgg tatcaacagg gacaccagga
     3001 tttatttatt ctgcgaagtg atcttccgtc acaggtattt attcggcgca aagtgcgtcg
     3061 ggtgatgctg ccaacttact gatttagtgt atgatggtgt ttttgaggtg ctccagtggc
     3121 ttctgtttct atcagggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     3181 gcccacccca aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     3241 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3301 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3361 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     3421 agcagagcgc agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc
     3481 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     3541 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     3601 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     3661 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     3721 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     3781 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     3841 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     3901 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     3961 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4021 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata
     4081 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     4141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag
     4201 gcaccccagg ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga
     4261 taacaatttc acacaggaaa cagctatgac atgattacga attcgagctc ggtacccggg
     4321 gatcctctag agtcgacctg caggcatgca agcttggcac tggccgtcgt tttacaacgt
     4381 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     4441 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     4501 ctgaatggcg aatggcgata agctagcttc acgctgccgc aagcactcag ggcgcaaggg
     4561 ctgctaaagg aagcggaaca cgtagaaagc cagtccgcag aaacggtgct gaccccggat
     4621 gaatgtcagc tactgggcta tctggacaag ggaaaacgca agcgcaaaga gaaagcaggt
     4681 agcttgcagt gggcttacat ggcgatagct agactgggcg gttttatgga cagcaagcga
     4741 accggaattg ccagctgggg cgccctctgg taaggttggg aagccctgca aagtaaactg
     4801 gatggctttc ttgccgccaa ggatctgatg gcgcagggga tcaagatctc cgaaaagtgc
     4861 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     4921 cgaggccctt tcgtcttcaa gaattctcat gtttgacagc ttatcatcga taagctttaa
     4981 tgcggtagtt tatcacagtt aaattgctaa cgcagtcagg caccgtgtat gaaatctaac
     5041 aatgcgctca tcgtcatcct cggcaccgtc accctggatg ctgtaggcat aggcttggtt
     5101 atgccggtac tgccgggcct cttgcgggat atcgtccatt ccgacagcat cgccagtcac
     5161 tatggcgtgc tgctagcgct atatgcgttg atgcaatttc tatgcgcacc cgttctcgga
     5221 gcactgtccg accgctttgg ccgccgccca gtcctgctcg cttcgctact tggagccact
     5281 atcgactacg cgatcatggc gaccacaccc gtcctgtgga tcctctacgc cggacgcatc
     5341 gtggccggca tcaccggcgc cacaggtgcg gttgctggcg cctatatcgc cgacatcacc
     5401 gatggggaag atcgggctcg ccacttcggg ctcatgagcg cttgtttcgg cgtgggtatg
     5461 gtggcaggcc ccgtggccgg gggactgttg ggcgccatct ccttgcatgc accattcctt
     5521 gcggcggcgg tgctcaacgg cctcaaccta ctactgggct gcttcctaat gcaggagtcg
     5581 cataagggag agcgtcgacc gatgcccttg agagccttca acccagtcag ctccttccgg
     5641 tgggcgcggg gcatgactat cgtcgccgca cttatgactg tcttctttat catgcaactc
     5701 gtaggacagg tgccggcagc gctctgggtc attttcggcg aggaccgctt tcgctggagc
     5761 gcgacgatga tcggcctgtc gcttgcggta ttcggaatct tgcacgccct cgctcaagcc
     5821 ttcgtcactg gtcccgccac caaacgtttc ggcgagaagc aggccattat cgccggcatg
     5881 gcggccgacg cgctgggcta cgtcttgctg gcgttcgcga cgcgaggctg gatggccttc
     5941 cccattatga ttcttctcgc ttccggcggc atcgggatgc ccgcgttgca ggccatgctg
     6001 tccaggcagg tagatgacga ccatcaggga cagcttcaag gatcgctcgc ggctcttacc
     6061 agcctaactt cgatcattgg accgctgatc gtcacggcga tttatgccgc ctcggcgagc
     6121 acatggaacg ggttggcatg gattgtaggc gccgccctat accttgtctg cctccccgcg
     6181 ttgcgtcgcg gtgcatggag ccgggccacc tcgacctgaa tggaagccgg cggcacctcg
     6241 ctaacggatt caccactcca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     6301 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     6361 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     6421 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     6481 gagttcttct gagcgggact ctggggttcg ctagaggatc gatccttttt aacccat
//
