LOCUS       sequence-139809-        8267 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Gateway-compatible 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 8267)
  TITLE     Kvitko lab Gateway compatible allelic exchange plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8267)
  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            "Unpublished"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: pDEST2T18ms
FEATURES             Location/Qualifiers
     source          1..8267
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     374..392
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(419..424,425..441,442..447)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             495..1685
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSITGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(847..866)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1828..1847
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     promoter        2004..2449
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(2450..2536,2537..3871)
                     /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            4225..4334
                     /note="incP origin of transfer"
     promoter        complement(4569..4659)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     promoter        complement(4853..4943)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      5303..5891
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5792..5811
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6045..6062
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6179..6200
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6215..6220,6221..6238,6239..6245)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6253..6269
                     /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     6258..6280
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6277..6293
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6277..6293
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    6331..6455
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(6496..6801)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(6598..6617)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(7143..7802)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYLPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     7717..7736
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(join(7856..7862,7863..7880,7881..7886))
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     protein_bind    complement(7911..8035)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(8092..8109)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(8092..8108)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(8101..8123)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 tgccgcaagc actcagggcg caagggctgc taaaggaagc ggaacacgta gaaagccagt
       61 ccgcagaaac ggtgctgacc ccggatgaat gtcagctact gggctatctg gacaagggaa
      121 aacgcaagcg caaagagaaa gcaggtagct tgcagtgggc ttacatggcg atagctagac
      181 tgggcggttt tatggacagc aagcgaaccg gaattgccag ctggggcgcc ctctggtaag
      241 gttgggaagc cctgcaaagt aaactggatg gctttcttgc cgccaaggat ctgatggcgc
      301 aggggatcaa gatctccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac
      361 attaacctat aaaaataggc gtatcacgag gccctttcgt cttcaagaat tctcatgttt
      421 gacagcttat catcgataag ctttaatgcg gtagtttatc acagttaaat tgctaacgca
      481 gtcaggcacc gtgtatgaaa tctaacaatg cgctcatcgt catcctcggc accgtcaccc
      541 tggatgctgt aggcataggc ttggttatgc cggtactgcc gggcctcttg cgggatatcg
      601 tccattccga cagcatcgcc agtcactatg gcgtgctgct agcgctatat gcgttgatgc
      661 aatttctatg cgcacccgtt ctcggagcac tgtccgaccg ctttggccgc cgcccagtcc
      721 tgctcgcttc gctacttgga gccactatcg actacgcgat catggcgacc acacccgtcc
      781 tgtggatcct ctacgccgga cgcatcgtgg ccggcatcac cggcgccaca ggtgcggttg
      841 ctggcgccta tatcgccgac atcaccgatg gggaagatcg ggctcgccac ttcgggctca
      901 tgagcgcttg tttcggcgtg ggtatggtgg caggccccgt ggccggggga ctgttgggcg
      961 ccatctcctt gcatgcacca ttccttgcgg cggcggtgct caacggcctc aacctactac
     1021 tgggctgctt cctaatgcag gagtcgcata agggagagcg tcgaccgatg cccttgagag
     1081 ccttcaaccc agtcagctcc ttccggtggg cgcggggcat gactatcgtc gccgcactta
     1141 tgactgtctt ctttatcatg caactcgtag gacaggtgcc ggcagcgctc tgggtcattt
     1201 tcggcgagga ccgctttcgc tggagcgcga cgatgatcgg cctgtcgctt gcggtattcg
     1261 gaatcttgca cgccctcgct caagccttcg tcactggtcc cgccaccaaa cgtttcggcg
     1321 agaagcaggc cattatcgcc ggcatggcgg ccgacgcgct gggctacgtc ttgctggcgt
     1381 tcgcgacgcg aggctggatg gccttcccca ttatgattct tctcgcttcc ggcggcatcg
     1441 ggatgcccgc gttgcaggcc atgctgtcca ggcaggtaga tgacgaccat cagggacagc
     1501 ttcaaggatc gctcgcggct cttaccagcc taacttcgat cactggaccg ctgatcgtca
     1561 cggcgattta tgccgcctcg gcgagcacat ggaacgggtt ggcatggatt gtaggcgccg
     1621 ccctatacct tgtctgcctc cccgcgttgc gtcgcggtgc atggagccgg gccacctcga
     1681 cctgaatgga agccggcggc acctcgctaa cggattcacc actccatggc gatgcctgct
     1741 tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt ggccggctgg
     1801 gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct gaagagcttg
     1861 gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc gattcgcagc
     1921 gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg ggttcgctag
     1981 aggatcgatc ctttttaacc catcacatat acctgccgtt cactattatt tagtgaaatg
     2041 agatattatg atattttctg aattgtgatt aaaaaggcaa ctttatgccc atgcaacaga
     2101 aactataaaa aatacagaga atgaaaagaa acagatagat tttttagttc tttaggcccg
     2161 tagtctgcaa atccttttat gattttctat caaacaaaag aggaaaatag accagttgca
     2221 atccaaacga gagtctaata gaatgaggtc gaaaagtaaa tcgcgcgggt ttgttactga
     2281 taaagcaggc aagacctaaa atgtgtaaag ggcaaagtgt atactttggc gtcacccctt
     2341 acatatttta ggtctttttt tattgtgcgt aactaacttg ccatcttcaa acaggagggc
     2401 tggaagaagc agaccgctaa cacagtacat aaaaaaggag acatgaacga tgaacatcaa
     2461 aaagtttgca aaacaagcaa cagtattaac ctttactacc gcactgctgg caggaggcgc
     2521 aactcaagcg tttgcgaaag aaacgaacca aaagccatat aaggaaacat acggcatttc
     2581 ccatattaca cgccatgata tgctgcaaat ccctgaacag caaaaaaatg aaaaatatca
     2641 agtttctgaa tttgattcgt ccacaattaa aaatatctct tctgcaaaag gcctggacgt
     2701 ttgggacagc tggccattac aaaacgctga cggcactgtc gcaaactatc acggctacca
     2761 catcgtcttt gcattagccg gagatcctaa aaatgcggat gacacatcga tttacatgtt
     2821 ctatcaaaaa gtcggcgaaa cttctattga cagctggaaa aacgctggcc gcgtctttaa
     2881 agacagcgac aaattcgatg caaatgattc tatcctaaaa gaccaaacac aagaatggtc
     2941 aggttcagcc acatttacat ctgacggaaa aatccgttta ttctacactg atttctccgg
     3001 taaacattac ggcaaacaaa cactgacaac tgcacaagtt aacgtatcag catcagacag
     3061 ctctttgaac atcaacggtg tagaggatta taaatcaatc tttgacggtg acggaaaaac
     3121 gtatcaaaat gtacagcagt tcatcgatga aggcaactac agctcaggcg acaaccatac
     3181 gctgagagat cctcactacg tagaagataa aggccacaaa tacttagtat ttgaagcaaa
     3241 cactggaact gaagatggct accaaggcga agaatcttta tttaacaaag catactatgg
     3301 caaaagcaca tcattcttcc gtcaagaaag tcaaaaactt ctgcaaagcg ataaaaaacg
     3361 cacggctgag ttagcaaacg gcgctctcgg tatgattgag ctaaacgatg attacacact
     3421 gaaaaaagtg atgaaaccgc tgattgcatc taacacagta acagatgaaa ttgaacgcgc
     3481 gaacgtcttt aaaatgaacg gcaaatggta cctgttcact gactcccgcg gatcaaaaat
     3541 gacgattgac ggcattacgt ctaacgatat ttacatgctt ggttatgttt ctaattcttt
     3601 aactggccca tacaagccgc tgaacaaaac tggccttgtg ttaaaaatgg atcttgatcc
     3661 taacgatgta acctttactt actcacactt cgctgtacct caagcgaaag gaaacaatgt
     3721 cgtgattaca agctatatga caaacagagg attctacgca gacaaacaat caacgtttgc
     3781 gccgagcttc ctgctgaaca tcaaaggcaa gaaaacatct gttgtcaaag acagcatcct
     3841 tgaacaagga caattaacag ttaacaaata aaaacgcaaa agaaaatgcc gatgggtacc
     3901 gagcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     3961 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccctcg cggacgtgct
     4021 catagtccac gacgcccgtg attttgtagc cctggccgac ggccagcagg taggccgaca
     4081 ggctcatgcc ggccgccgcc gccttttcct caatcgctct tcgttcgtct ggaaggcagt
     4141 acaccttgat aggtgggctg cccttcctgg ttggcttggt ttcatcagcc atccgcttgc
     4201 cctcatctgt tacgccggcg gtagccggcc agcctcgcag agcaggattc ccgttgagca
     4261 ccgccaggtg cgaataaggg acagtgaaga aggaacaccc gctcgcgggt gggcctactt
     4321 cacctatcct gcccggctga cgccgttgga tacaccaagg aaagtctaca cgaacccttt
     4381 ggcaaaatcc tgtatatcgt gcgaaaaagg atggatatac cgaaaaaatc gctataatga
     4441 ccccgaagca gggttatgca gcggaaaagc gctgcttccc tgctgttttg tggaatatct
     4501 accgactgga aacaggcaaa tgcaggaaat tactgaactg aggggacagg cgagagacga
     4561 tgccaaagag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt
     4621 tcattatggt gaaagttgga acctcttacg tgccgatcaa cgtctcattt tcgccaaaag
     4681 ttggcccagg gcttcccggt atcaacaggg acaccaggat ttatttattc tgcgaagtga
     4741 tcttccgtca caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc caacttactg
     4801 atttagtgta tgatggtgtt tttgaggtgc tccagtggct tctgtttcta tcagctcctg
     4861 aaaatctcga taactcaaaa aatacgcccg gtagtgatct tatttcatta tggtgaaagt
     4921 tggaacctct tacgtgccga tcaacgtctc attttcgcca aaagttggcc cagggcttcc
     4981 cggtatcaac agggacacca ggatttattt attctgcgaa gtgatcttcc gtcacaggta
     5041 tttattcggc gcaaagtgcg tcgggtgatg ctgccaactt actgatttag tgtatgatgg
     5101 tgtttttgag gtgctccagt ggcttctgtt tctatcaggg ctggatgatc ctccagcgcg
     5161 gggatctcat gctggagttc ttcgcccacc ccaaaaggat ctaggtgaag atcctttttg
     5221 ataatctcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     5281 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     5341 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     5401 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     5461 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     5521 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     5581 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     5641 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     5701 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     5761 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     5821 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     5881 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     5941 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     6001 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     6061 aggaagcgga agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt
     6121 aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta
     6181 atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta
     6241 tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat gacatgatta
     6301 cgaattcgag ctcggtaccc ctccacatca accactttgt acaagaaagc tgaacgagaa
     6361 acgtaaaatg atataaatat caatatatta aattagattt tgcataaaaa acagactaca
     6421 taatactgta aaacacaaca tatccagtca ctatggtcga cctgcagact ggctgtgtat
     6481 aagggagcct gacatttata ttccccagaa catcaggtta atggcgtttt tgatgtcatt
     6541 ttcgcggtgg ctgagatcag ccacttcttc cccgataacg gagaccggca cactggccat
     6601 atcggtggtc atcatgcgcc agctttcatc cccgatatgc accaccgggt aaagttcacg
     6661 ggagacttta tctgacagca gacgtgcact ggccaggggg atcaccatcc gtcgcccggg
     6721 cgtgtcaata atatcactct gtacatccac aaacagacga taacggctct ctcttttata
     6781 ggtgtaaacc ttaaactgca tttcaccagc ccctgttctc gtcagcaaaa gagccgttca
     6841 tttcaataaa ccgggcgacc tcagccatcc cttcctgatt ttccgctttc cagcgttcgg
     6901 cacgcagacg acgggcttca ttctgcatgg ttgtgcttac cagaccggag atattgacat
     6961 catatatgcc ttgagcaact gatagctgtc gctgtcaact gtcactgtaa tacgctgctt
     7021 catagcacac ctctttttga catacttcgg gtatacatat cagtatatat tcttataccg
     7081 caaaaatcag cgcgcaaata cgcatactgt tatctggctt ttagtaagcc ggatccagat
     7141 ctttacgccc cgccctgcca ctcatcgcag tactgttgta attcattaag cattctgccg
     7201 acatggaagc catcacagac ggcatgatga acctgaatcg ccagcggcat cagcaccttg
     7261 tcgccttgcg tataatattt gcccatggtg aaaacggggg cgaagaagtt gtccatattg
     7321 gccacgttta aatcaaaact ggtgaaactc acccagggat tggctgagac gaaaaacata
     7381 ttctcaataa accctttagg gagataggcc aggttttcac cgtaacacgc cacatcttgc
     7441 gaatatatgt gtagaaactg ccggaaatcg tcgtggtatt cactccagag cgatgaaaac
     7501 gtttcagttt gctcatggaa aacggtgtaa caagggtgaa cactatccca tatcaccagc
     7561 tcaccgtctt tcattgccat acggaattcc ggatgagcat tcatcaggcg ggcaagaatg
     7621 tgaataaagg ccggataaaa cttgtgctta tttttcttta cggtctttaa aaaggccgta
     7681 atatccagct gaacggtctg gttataggta cattgagcaa ctgactgaaa tgcctcaaaa
     7741 tgttctttac gatgccattg ggatatatca acggtggtat atccagtgat ttttttctcc
     7801 attttagctt ccttagctcc tgaaaatctc gacggatcct aactcaaaat ccacacatta
     7861 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgcggccgc catagtgact
     7921 ggatatgttg tgttttacag tattatgtag tctgtttttt atgcaaaatc taatttaata
     7981 tattgatatt tatatcattt tacgtttctc gttcagcttt tttgtacaaa cttgtgcatg
     8041 catacccgag ggggatcctc tagagtcgac ctgcaggcat gcaagcttgg cactggccgt
     8101 cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc gccttgcagc
     8161 acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca
     8221 acagttgcgc agcctgaatg gcgaatggcg ataagctagc ttcacgc
//
