LOCUS       sequence-139808-        7454 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 7454)
  AUTHORS   Mijatovic J, Severns PM, Kemerait RC, Walcott RR, Kvitko BH
  TITLE     Patterns of Seed-to-Seedling Transmission of Xanthomonas citri pv. 
            malvacearum, the Causal Agent of Cotton Bacterial Blight.
  JOURNAL   Phytopathology. 2021 Dec;111(12):2176-2184. doi: 
            10.1094/PHYTO-02-21-0057-R. Epub 2021 Nov 22.
  PUBMED    34032522
REFERENCE   2  (bases 1 to 7454)
  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"; doi: 
            "10.1094/PHYTO-02-21-0057-R"; journalName: "Phytopathology"; date: 
            "2021-12"; volume: "111"; issue: "12"; pages: "2176-2184"
COMMENT     Sequence Label: pDEST1K18ms
FEATURES             Location/Qualifiers
     source          1..7454
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             346..1140
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(400..419)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1010..1029
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     promoter        1186..1631
                     /gene="sacR"
                     /label=sacB promoter
                     /note="sacB promoter and control region"
     CDS             join(1632..1718,1719..3053)
                     /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            3407..3516
                     /note="incP origin of transfer"
     promoter        complement(3751..3841)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     promoter        complement(4035..4125)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     rep_origin      4485..5073
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4974..4993
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5227..5244
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5361..5382
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5397..5402,5403..5420,5421..5427)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5435..5451
                     /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     5440..5462
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5459..5475
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5459..5475
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     protein_bind    5518..5642
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(5667..5672,5673..5690,5691..5697)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             5751..6410
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(5817..5836)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             6752..7057
                     /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     6936..6955
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(7098..7222)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(7279..7296)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7279..7295)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7288..7310)
                     /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 gatctgatca agagacagga tgaggatcgt ttcgcatgat tgaacaagat
      361 ggattgcacg caggttctcc ggccgcttgg gtggagaggc tattcggcta tgactgggca
      421 caacagacaa tcggctgctc tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg
      481 gttctttttg tcaagaccga cctgtccggt gccctgaatg aactccaaga cgaggcagcg
      541 cggctatcgt ggctggccac gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact
      601 gaagcgggaa gggactggct gctattgggc gaagtgccgg ggcaggatct cctgtcatct
      661 caccttgctc ctgccgagaa agtatccatc atggctgatg caatgcggcg gctgcatacg
      721 cttgatccgg ctacctgccc attcgaccac caagcgaaac atcgcatcga gcgagcacgt
      781 actcggatgg aagccggtct tgtcgatcag gatgatctgg acgaagagca tcaggggctc
      841 gcgccagccg aactgttcgc caggctcaag gcgcggatgc ccgacggcga ggatctcgtc
      901 gtgacccatg gcgatgcctg cttgccgaat atcatggtgg aaaatggccg cttttctgga
      961 ttcatcgact gtggccggct gggtgtggcg gaccgctatc aggacatagc gttggctacc
     1021 cgtgatattg ctgaagagct tggcggcgaa tgggctgacc gcttcctcgt gctttacggt
     1081 atcgccgctc ccgattcgca gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga
     1141 gcgggactct ggggttcgct agaggatcga tcctttttaa cccatcacat atacctgccg
     1201 ttcactatta tttagtgaaa tgagatatta tgatattttc tgaattgtga ttaaaaaggc
     1261 aactttatgc ccatgcaaca gaaactataa aaaatacaga gaatgaaaag aaacagatag
     1321 attttttagt tctttaggcc cgtagtctgc aaatcctttt atgattttct atcaaacaaa
     1381 agaggaaaat agaccagttg caatccaaac gagagtctaa tagaatgagg tcgaaaagta
     1441 aatcgcgcgg gtttgttact gataaagcag gcaagaccta aaatgtgtaa agggcaaagt
     1501 gtatactttg gcgtcacccc ttacatattt taggtctttt tttattgtgc gtaactaact
     1561 tgccatcttc aaacaggagg gctggaagaa gcagaccgct aacacagtac ataaaaaagg
     1621 agacatgaac gatgaacatc aaaaagtttg caaaacaagc aacagtatta acctttacta
     1681 ccgcactgct ggcaggaggc gcaactcaag cgtttgcgaa agaaacgaac caaaagccat
     1741 ataaggaaac atacggcatt tcccatatta cacgccatga tatgctgcaa atccctgaac
     1801 agcaaaaaaa tgaaaaatat caagtttctg aatttgattc gtccacaatt aaaaatatct
     1861 cttctgcaaa aggcctggac gtttgggaca gctggccatt acaaaacgct gacggcactg
     1921 tcgcaaacta tcacggctac cacatcgtct ttgcattagc cggagatcct aaaaatgcgg
     1981 atgacacatc gatttacatg ttctatcaaa aagtcggcga aacttctatt gacagctgga
     2041 aaaacgctgg ccgcgtcttt aaagacagcg acaaattcga tgcaaatgat tctatcctaa
     2101 aagaccaaac acaagaatgg tcaggttcag ccacatttac atctgacgga aaaatccgtt
     2161 tattctacac tgatttctcc ggtaaacatt acggcaaaca aacactgaca actgcacaag
     2221 ttaacgtatc agcatcagac agctctttga acatcaacgg tgtagaggat tataaatcaa
     2281 tctttgacgg tgacggaaaa acgtatcaaa atgtacagca gttcatcgat gaaggcaact
     2341 acagctcagg cgacaaccat acgctgagag atcctcacta cgtagaagat aaaggccaca
     2401 aatacttagt atttgaagca aacactggaa ctgaagatgg ctaccaaggc gaagaatctt
     2461 tatttaacaa agcatactat ggcaaaagca catcattctt ccgtcaagaa agtcaaaaac
     2521 ttctgcaaag cgataaaaaa cgcacggctg agttagcaaa cggcgctctc ggtatgattg
     2581 agctaaacga tgattacaca ctgaaaaaag tgatgaaacc gctgattgca tctaacacag
     2641 taacagatga aattgaacgc gcgaacgtct ttaaaatgaa cggcaaatgg tacctgttca
     2701 ctgactcccg cggatcaaaa atgacgattg acggcattac gtctaacgat atttacatgc
     2761 ttggttatgt ttctaattct ttaactggcc catacaagcc gctgaacaaa actggccttg
     2821 tgttaaaaat ggatcttgat cctaacgatg taacctttac ttactcacac ttcgctgtac
     2881 ctcaagcgaa aggaaacaat gtcgtgatta caagctatat gacaaacaga ggattctacg
     2941 cagacaaaca atcaacgttt gcgccgagct tcctgctgaa catcaaaggc aagaaaacat
     3001 ctgttgtcaa agacagcatc cttgaacaag gacaattaac agttaacaaa taaaaacgca
     3061 aaagaaaatg ccgatgggta ccgagcgaaa tgaccgacca agcgacgccc aacctgccat
     3121 cacgagattt cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc
     3181 gggacgccct cgcggacgtg ctcatagtcc acgacgcccg tgattttgta gccctggccg
     3241 acggccagca ggtaggccga caggctcatg ccggccgccg ccgccttttc ctcaatcgct
     3301 cttcgttcgt ctggaaggca gtacaccttg ataggtgggc tgcccttcct ggttggcttg
     3361 gtttcatcag ccatccgctt gccctcatct gttacgccgg cggtagccgg ccagcctcgc
     3421 agagcaggat tcccgttgag caccgccagg tgcgaataag ggacagtgaa gaaggaacac
     3481 ccgctcgcgg gtgggcctac ttcacctatc ctgcccggct gacgccgttg gatacaccaa
     3541 ggaaagtcta cacgaaccct ttggcaaaat cctgtatatc gtgcgaaaaa ggatggatat
     3601 accgaaaaaa tcgctataat gaccccgaag cagggttatg cagcggaaaa gcgctgcttc
     3661 cctgctgttt tgtggaatat ctaccgactg gaaacaggca aatgcaggaa attactgaac
     3721 tgaggggaca ggcgagagac gatgccaaag agctcctgaa aatctcgata actcaaaaaa
     3781 tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc
     3841 aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg
     3901 atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc
     3961 gggtgatgct gccaacttac tgatttagtg tatgatggtg tttttgaggt gctccagtgg
     4021 cttctgtttc tatcagctcc tgaaaatctc gataactcaa aaaatacgcc cggtagtgat
     4081 cttatttcat tatggtgaaa gttggaacct cttacgtgcc gatcaacgtc tcattttcgc
     4141 caaaagttgg cccagggctt cccggtatca acagggacac caggatttat ttattctgcg
     4201 aagtgatctt ccgtcacagg tatttattcg gcgcaaagtg cgtcgggtga tgctgccaac
     4261 ttactgattt agtgtatgat ggtgtttttg aggtgctcca gtggcttctg tttctatcag
     4321 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaaaagg
     4381 atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg
     4441 ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt
     4501 ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg
     4561 ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata
     4621 ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca
     4681 ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag
     4741 tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc
     4801 tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga
     4861 tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg
     4921 tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac
     4981 gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg
     5041 tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg
     5101 ttcctggcct tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct
     5161 gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc
     5221 gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc caatacgcaa accgcctctc
     5281 cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg
     5341 ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc ccaggcttta
     5401 cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca atttcacaca
     5461 ggaaacagct atgacatgat tacgaattcg agctcggtac ccctcgggta tgcatgcaca
     5521 agtttgtaca aaaaagctga acgagaaacg taaaatgata taaatatcaa tatattaaat
     5581 tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat ccagtcacta
     5641 tggcggccgc attaggcacc ccaggcttta cactttatgc ttccggctcg tataatgtgt
     5701 ggattttgag ttaggatccg gcgagatttt caggagctaa ggaagctaaa atggagaaaa
     5761 aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg
     5821 catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct
     5881 ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg
     5941 cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga
     6001 tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat
     6061 cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg
     6121 tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt
     6181 tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg
     6241 acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc
     6301 tgatgccgct ggcgattcag gttcatcatg ccgtctgtga tggcttccat gtcggcagaa
     6361 tgcttaatga attacaacag tactgcgatg agtggcaggg cggggcgtaa agatctggat
     6421 ccggcttact aaaagccaga taacagtatg cgtatttgcg cgctgatttt tgcggtataa
     6481 gaatatatac tgatatgtat acccgaagta tgtcaaaaag aggtatgcta tgaagcagcg
     6541 tattacagtg acagttgaca gcgacagcta tcagttgctc aaggcatata tgatgtcaat
     6601 atctccggtc tggtaagcac aaccatgcag aatgaagccc gtcgtctgcg tgccgaacgc
     6661 tggaaagcgg aaaatcagga agggatggct gaggtcgccc ggtttattga aatgaacggc
     6721 tcttttgctg acgagaacag gggctggtga aatgcagttt aaggtttaca cctataaaag
     6781 agagagccgt tatcgtctgt ttgtggatgt acagagtgat attattgaca cgcccgggcg
     6841 acggatggtg atccccctgg ccagtgcacg tctgctgtca gataaagtct cccgtgaact
     6901 ttacccggtg gtgcatatcg gggatgaaag ctggcgcatg atgaccaccg atatggccag
     6961 tgtgccggtc tccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat
     7021 caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggctccc ttatacacag
     7081 ccagtctgca ggtcgaccat agtgactgga tatgttgtgt cttacagtat tatgtagtct
     7141 gttttttatg caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt
     7201 cagctttttt gtacaaactt gtgcatgcat acccgagggg gatcctctag agtcgacctg
     7261 caggcatgca agcttggcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     7321 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     7381 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgata
     7441 agctagcttc acgc
//
