LOCUS       sequence-220090-        9635 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Broad host range replicative plasmid expressing the I-SceI homing 
            endonuclease and the counterselectable marker SacB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9635)
  AUTHORS   Aubert DF, Hamad MA, Valvano MA
  TITLE     A markerless deletion method for genetic manipulation of 
            Burkholderia cenocepacia and other multidrug-resistant gram-negative
            bacteria.
  JOURNAL   Methods Mol Biol. 2014;1197:311-27. doi: 
            10.1007/978-1-4939-1261-2_18.
  PUBMED    25172289
REFERENCE   2  (bases 1 to 9635)
  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: "Methods Mol
            Biol."; date: "2014"; volume: "1197:311-27. doi"; pages: " 
            10.1007/978-1-4939-1261-2_18"
COMMENT     Sequence Label: pDAI-SceI-SacB
FEATURES             Location/Qualifiers
     source          1..9635
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(1574..1593)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     CDS             complement(1694..2344)
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline resistance regulatory protein"
                     /label=TetR
                     /translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
                     RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
                     PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
                     QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGDD"
     CDS             2450..3649
                     /codon_start=1
                     /gene="tetA"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKPNIPLIVILSTVALDAVGIGLIMPVLPGLLRDLVHSNDVTAHY
                     GILLALYALVQFACAPVLGALSDRFGRRPILLVSLAGATVDYAIMATAPFLWVLYIGRI
                     VAGITGATGAVAGAYIADITDGDERARHFGFMSACFGFGMVAGPVLGGLMGGFSPHAPF
                     FAAAALNGLNFLTGCFLLPESHKGERRPLRREALNPLASFRWARGMTVVAALMAVFFIM
                     QLVGQVPAALWVIFGEDRFHWDATTIGISLAAFGILHSLAQAMITGPVAARLGERRALM
                     LGMIADGTGYILLAFATRGWMAFPIMVLLASGGIGMPALQAMLSRQVDEERQGQLQGSL
                     AALTSLTSIVGPLLFTAIYAASITTWNGWAWIAGAALYLLCLPALRRGLWSGAGQRADR
                     "
     CDS             complement(4177..4839)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQRRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      4840..5611
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     primer_bind     6869..6887
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        7004..7106
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(7173..7192)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      complement(7960..7987)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(8079..8165)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     8318..8335
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     CDS             complement(8393..8407)
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     promoter        complement(join(9462..9467,9468..9484,9485..9490))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     protein_bind    9599..9615
                     /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)."
ORIGIN
        1 atgacgtact ccaaacaagc cgtttcccgt agacacaaac gtttggccct gagcgccgcc
       61 gcgctcaccg cagccgcttg catgtcggcg cacgcgcaga gcgacggcgc aactcaagcg
      121 tttgcggaag aaacgaacca aaagccatat aaggaaacat acggcatttc ccatattaca
      181 cgccatgata tgctgcaaat ccctgaacag caaaaaaatg aaaaatatca agtttctgaa
      241 tttgattcgt ccacaattaa aaatatctct tctgcaaaag gcctggacgt ttgggacagc
      301 tggccattac aaaacgctga cggcactgtc gcaaactatc acggctacca catcgtcttt
      361 gcattagccg gagatcctaa aaatgcggat gacacatcga tttacatgtt ctatcaaaaa
      421 gtcggcgaaa cttctattga cagctggaaa aacgctggcc gcgtctttaa agacagcgac
      481 aaattcgatg caaatgattc tatcctaaaa gaccaaacac aagaatggtc aggttcagcc
      541 acatttacat ctgacggaaa aatccgttta ttctacactg atttctccgg taaacattac
      601 ggcaaacaaa cactgacaac tgcacaagtt aacgtatcag catcagacag ctctttgaac
      661 atcaacggtg tagaggatta taaatcaatc tttgacggtg acggaaaaac gtatcaaaat
      721 gtacagcagt tcatcgatga aggcaactac agctcaggcg acaaccatac gctgagagat
      781 cctcactacg tagaagataa aggccacaaa tacttagtat ttgaagcaaa cactggaact
      841 gaagatggct accaaggcga agaatcttta tttaacaaag catactatgg caaaagcaca
      901 tcattcttcc gtcaagaaag tcaaaaactt ctgcaaagcg ataaaaaacg cccggctgag
      961 ttagcaaacg gcgctctcgg tatgattgag ctaaacgatg attacacact gaaaaaagtg
     1021 atgaaaccgc tgattgcatc taacacagta acagatgaaa ttgaacgcgc gaacgtcttt
     1081 aaaatgaacg gcaaatggta cctgttcact gactcccgcg gatcaaaaat gacgattgac
     1141 ggcattacgt ctaacgatat ttacatgctt ggttatgttt ctaattcttt aactggccca
     1201 tacaagccgc tgaacaaaac tggccttgtg ttaaaaatgg atcttgatcc taacgatgta
     1261 acctttactt actcacactt cgctgtacct caagcgaaag gaaacaatgt cgtgattaca
     1321 agctatatga caaacagagg attctacgca gacaaacaat caacgtttgc gccgagcttc
     1381 ctgctgaaca tcaaaggcaa gaaaacatct gttgtcaaag acagcatcct tgaacaagga
     1441 caattaacag ttaacaaata actgcagctt gctcgagcta gcatactcga gtaagctgtc
     1501 aaacatgagc agatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt
     1561 gcggttgctg gcgcctatat cgccgacatc accgatggga gatcctaaga tatcgcttag
     1621 gccacacgtt caagtgcaga tcgatcgatg gctctgctgt agtgagtggg ttgcgctccg
     1681 gcagcggtcc tgatcaatcg tcaccctttc tcggtccttc aacgttcctg acaacgagcc
     1741 tccttttcgc caatccatcg acaatcaccg cgagtccctg ctcgaacgct gcgtccggac
     1801 cggcttcgtc gaaggcgtct atcgcggccc gcaacagcgg cgagagcgga gcctgttcaa
     1861 cggtgccgcc gcgctcgccg gcatcgctgt cgccggcctg ctcctcaagc acggccccaa
     1921 cagtgaagta gctgattgtc atcagcgcat tgacggcgtc cccggccgaa aaacccgcct
     1981 cgcagaggaa gcgaagctgc gcgtcggccg tttccatctg cggtgcgccc ggtcgcgtgc
     2041 cggcatggat gcgcgcgcca tcgcggtagg cgagcagcgc ctgcctgaag ctgcgggcat
     2101 tcccgatcag aaatgagcgc cagtcgtcgt cggctctcgg caccgaatgc gtatgattct
     2161 ccgccagcat ggcttcggcc agtgcgtcga gcagcgcccg cttgttcctg aagtgccagt
     2221 aaagcgccgg ctgctgaacc cccaaccgtt ccgccagttt gcgtgtcgtc agaccgtcta
     2281 cgccgacctc gttcaacagg tccagggcgg cacggatcac tgtattcggc tgcaactttg
     2341 tcatgcttga cactttatca ctgataaaca taatatgtcc accaacttat cagtgataaa
     2401 gaatccgcgc gttcaatcgg accagcggag gctggtccgg aggccagacg tgaaacccaa
     2461 catacccctg atcgtaattc tgagcactgt cgcgctcgac gctgtcggca tcggcctgat
     2521 tatgccggtg ctgccgggcc tcctgcgcga tctggttcac tcgaacgacg tcaccgccca
     2581 ctatggcatt ctgctggcgc tgtatgcgtt ggtgcaattt gcctgcgcac ctgtgctggg
     2641 cgcgctgtcg gatcgtttcg ggcggcggcc aatcttgctc gtctcgctgg ccggcgccac
     2701 tgtcgactac gccatcatgg cgacagcgcc tttcctttgg gttctctata tcgggcggat
     2761 cgtggccggc atcaccgggg cgactggggc ggtagccggc gcttatattg ccgatatcac
     2821 tgatggcgat gagcgcgcgc ggcacttcgg cttcatgagc gcctgtttcg ggttcgggat
     2881 ggtcgcggga cctgtgctcg gtgggctgat gggcggtttc tccccccacg ctccgttctt
     2941 cgccgcggca gccttgaacg gcctcaattt cctgacgggc tgtttccttt tgccggagtc
     3001 gcacaaaggc gaacgccggc cgttacgccg ggaggctctc aacccgctcg cttcgttccg
     3061 gtgggcccgg ggcatgaccg tcgtcgccgc cctgatggcg gtcttcttca tcatgcaact
     3121 tgtcggacag gtgccggccg cgctttgggt cattttcggc gaggatcgct ttcactggga
     3181 cgcgaccacg atcggcattt cgcttgccgc atttggcatt ctgcattcac tcgcccaggc
     3241 aatgatcacc ggccctgtag ccgcccggct cggcgaaagg cgggcactca tgctcggaat
     3301 gattgccgac ggcacaggct acatcctgct tgccttcgcg acacggggat ggatggcgtt
     3361 cccgatcatg gtcctgcttg cttcgggtgg catcggaatg ccggcgctgc aagcaatgtt
     3421 gtccaggcag gtggatgagg aacgtcaggg gcagctgcaa ggctcactgg cggcgctcac
     3481 cagcctgacc tcgatcgtcg gacccctcct cttcacggcg atctatgcgg cttctataac
     3541 aacgtggaac gggtgggcat ggattgcagg cgctgccctc tacttgctct gcctgccggc
     3601 gctgcgtcgc gggctttgga gcggcgcagg gcaacgagcc gatcgctgat cgtggaaacg
     3661 ataggaaacg tggccaatat ggacaacttc ttcgcatcga tttcgttcca ctgagcgtca
     3721 gaccccgtag gttattggtc gacttaaacg cctggtgcta cgcctgaata agtgataata
     3781 agcggatgaa tggcagaaat tcgaaagcaa attcgacccg gtcgtcggtt cagggcaggg
     3841 tcgttaaata gccgcttatg tctattgctg gtttaccggt ttattgacta ccggaagcag
     3901 tgtgaccgtg tgcttctcaa atgcctgagg ccagtttgct caggctctcc ccgtggaggt
     3961 aataattgac gatatgatca tttattctgc ctcccagagc ctgataaaaa cggtgaatcc
     4021 gttagcgagg tgccgccggc ttccattcag gtcgaggtgg cccggctcca tgcaccgcga
     4081 cgcaacgcgg ggaggcagac aaggtatagg gcggcgaggc ggctacagcc gatagtctgg
     4141 aacagcgcac ttacgggttg ctgcgcaacc caagtgctac cggcgcggca gcgtgacccg
     4201 tgtcggcggc tccaacggct cgccatcgtc cagaaaacac ggctcatcgg gcatcggcag
     4261 gcgccgctgc ccgcgccgtt cccattcctc cgtttcggtc aaggctggca ggtctggttc
     4321 catgcccgga atgccgggct ggctgggcgg ctcctcgccg gggccggtcg gtagttgctg
     4381 ctcgcccgga tacagggtcg ggatgcggcg caggtcgcca tgccccaaca gcgattcgtc
     4441 ctggtcgtcg tgatcaacca ccacggcggc actgaacacc gacaggcgca actggtcgcg
     4501 gggctggccc cacgccacgc ggtcattgac cacgtaggcc gacacggtgc cggggccgtt
     4561 gagcttcacg acggagatcc agcgctcggc caccaagtcc ttgactgcgt attggaccgt
     4621 ccgcaaagaa cgtccgatga gcttggaaag tgtcttctgg ctgaccacca cggcgttctg
     4681 gtggcccatc tgcgccacga ggtgatgcag cagcattgcc gccgtgggtt tcctcgcaat
     4741 aagcccggcc cacgcctcat gcgctttgcg ttccgtttgc acccagtgac cgggcttgtt
     4801 cttggcttga atgccgattt ctctggactg cgtggccatg cttatctcca tgcggtaggg
     4861 gtgccgcacg gttgcggcac catgcgcaat cagctgcaac ttttcggcag cgcgacaaca
     4921 attatgcgtt gcgtaaaagt ggcagtcaat tacagatttt ctttaaccta cgcaatgagc
     4981 tattgcgggg ggtgccgcaa tgagctgttg cgtacccccc ttttttaagt tgttgatttt
     5041 taagtctttc gcatttcgcc ctatatctag ttctttggtg cccaaagaag ggcacccctg
     5101 cggggttccc ccacgccttc ggcgcggctc cccctccggc aaaaagtggc ccctccgggg
     5161 cttgttgatc gactgcgcgg ccttcggcct tgcccaaggt ggcgctgccc ccttggaacc
     5221 cccgcactcg ccgccgtgag gctcgggggg caggcgggcg ggcttcgccc ttcgactgcc
     5281 cccactcgca taggcttggg tcgttccagg cgcgtcaagg ccaagccgct gcgcggtcgc
     5341 tgcgcgagcc ttgacccgcc ttccacttgg tgtccaaccg gcaagcgaag cgcgcaggcc
     5401 gcaggccgga ggcttttccc cagagaaaat taaaaaaatt gatggggcaa ggccgcaggc
     5461 cgcgcagttg gagccggtgg gtatgtggtc gaaggctggg tagccggtgg gcaatccctg
     5521 tggtcaagct cgtgggcagg cgcagcctgt ccatcagctt gtccagcagg gttgtccacg
     5581 ggccgagcga agcgagccag ccggtggccg ctcgcggcca tcgtccacat atccacgggc
     5641 tggcaaggga gcgcagcgac cgcgcagggc gaagcccgga gagcaagccc gtagggcgcc
     5701 gcagccgccg taggcggtca cgactttgcg aagcaaagtc tagtgagtat actcaagcat
     5761 tgagtggccc gccggaggca ccgccttgcg ctgcccccgt cgagccggtt ggacaccaaa
     5821 agggaggggc aggcatggcg gcatacgcga tcatgcgatg caagaagctg gcgaaaatgg
     5881 gcaacgtggc ggccagtctc aagcacgcct accgcgagcg cgagacgccc aacgctgacg
     5941 ccagcaggac gccagagaac gagcactggg cggccagcag caccgatgaa gcgatgggcc
     6001 gactgcgcga gttgctgcca gagaagcggc gcaaggacgc tgtgttggcg gtcgagtacg
     6061 tcatgacggc cagcccggaa tggtggaagt cggccagcca agaacagcag gcggcgttct
     6121 tcgagaaggc gcacaagtgg ctggcggaca agtacggggc ggatcgcatc gtgacggcca
     6181 gcatccaccg tgacgaaacc agcccgcaca tgaccgcgtt cgtggtgccg ctgacgcagg
     6241 acggcaggct gtcggccaag gagttcatcg gcaacaaagc gcagatgacc cgcgaccaga
     6301 ccacgtttgc ggccgctgtg gccgatctag ggctgcaacg gggcatcgag ggcagcaagg
     6361 cacgtcacac gcgcattcag gcgttctacg aggccctgga gcggccacca gtgggccacg
     6421 tcaccatcag cccgcaagcg gtcgagccac gcgcctatgc accgcaggga ttggccgaaa
     6481 agctgggaat ctcaaagcgc gttgagacgc cggaagccgt ggccgaccgg ctgacaaaag
     6541 cggttcggca ggggtatgag cctgccctac aggccgccgc aggagcgcgt gagatgcgca
     6601 agaaggccga tcaagcccaa gagacggccc gagaccttcg ggagcgcctg aagcccgttc
     6661 tggacgccct ggggccgttg aatcgggata tgcaggccaa ggccgccgcg atcatcaagg
     6721 ccgtgggcga aaagctgctg acggaacagc gggaagtcca gcgccagaaa caggcccagc
     6781 gccagcagga acgcgggcgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     6841 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtcttcg
     6901 aataaatacc tgtgacggaa gatcacttcg cagaataaat aaatcctggt gtccctgttg
     6961 ataccgggaa gccctgggcc aacttttggc gaaaatgaga cgttgatcgg cacgtaagag
     7021 gttccaactt tcaccataat gaaataagat cactaccggg cgtatttttt gagttatcga
     7081 gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat accaccgttg
     7141 atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta
     7201 cctataacca gaccgttcag ctggatatta cggccttttt agcttttgcc attctcaccg
     7261 gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa
     7321 ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc
     7381 atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa
     7441 tacggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt
     7501 ttctaatcag aattggttaa ttggttgtaa cactggcaga gcattacgct gacttgacgg
     7561 gacggcggct ttgttgaata aatcgaactt ttgctgagtt gaaggatcag atcacgcatc
     7621 ttcccgacaa cgcagaccgt tccgtggcaa agcaaaagtt caaaatcacc aactggtcca
     7681 cctacaacaa agctctcatc aaccgtggct ccctcacttt ctggctggat gatggggcga
     7741 ttcaggcctg gtatgagtca gcaacacctt cttcacgagg cagacctcag cgccagaagg
     7801 ccgccagaga ggccgagcgc ggccgtgagg cttggacgct agggcagggc atgaaaaagc
     7861 ccgtagcggg ctgctacggg cgtctgacgc ggtggaaagg gggaggggat gttgtttaaa
     7921 tggctctgtg tagtgagtgg aagagtttgt agaaacgcaa aaaggccatc cgtcaggatg
     7981 gccttctgct taatttgatg cctggcagtt tatggcgggc gtcctgcccg ccaccctccg
     8041 ggccgttgct tcgcagcgtt caaatccgct cccggcggat ttgtcctact caggagagcg
     8101 ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc ctttcgtttt
     8161 atttgatgcc tggcagttcc ctactctcgc atggggagac cccacactac catcggcgct
     8221 acggcgtttc acttctgagt tcggcatggg gtcaggtggg accaccgcgc tactgccgcc
     8281 aggcaaattc tgttttatca gaccgcttct gcgttctgat ttaatctgta tcaggctgaa
     8341 aatcttctct catccgccaa aacagccaag cttgcatgcc tgcaggtcgt cacttgtcgt
     8401 cgtcgtcgtc cttgtagtct ctagattatt tcaggaaagt ttcggaggag atagtgttcg
     8461 gcagtttgta catcatctgc gggatcaggt acggtttgat caggttgtag aagatcaggt
     8521 aagacataga atcgatgtag atgatcggtt tgtttttgtt gatttttacg taacagttca
     8581 gttggaattt gttacgcaga cccttaacca ggtattctac ttcttcgaaa gtgaaagact
     8641 gggtgttcag tacgatcgat ttgttggtag agtttttgtt gtaatcccat ttaccaccat
     8701 catccatgaa ccagtatgcc agagacatcg gggtcaggta gttttcaacc aggttgttcg
     8761 ggatggtttt tttgttgtta acgatgaaca ggttagccag tttgttgaaa gcttggtgtt
     8821 tgaaagtctg ggcgccccag gtgattacca ggttacccag gtggttaaca cgttcttttt
     8881 tgtgcggcgg ggacagtacc cactgatcgt acagcagaca tacgtggtcc atgtatgctt
     8941 tgtttttcca ctcgaactgc atacagtagg ttttaccttc atcacgagaa cggatgtaag
     9001 catcacccag gatcagaccg atacctgctt cgaactgttc gatgttcagt tcgatcagct
     9061 gggatttgta ttctttcagc agtttagagt tcggacccag gttcattacc tggttttttt
     9121 gatgcatatg tagaattccg aatccttctt gtgaatctat tatggcgaca agcaaatcga
     9181 gctctgactg cctaccccac aacaactatc agaaagcacc agcacaacgg ctgcctaact
     9241 ttgttttagg gcgactgccc tgctgcgtaa catcgttgct gctccataac atcaaacatc
     9301 gacccacggc gtaacgcgct tgctgcttgg atgcccgagg catagactgt acaaaaaaac
     9361 agtcataaca agccatgaaa accgccactg cgccgttacc accgctgcgt tcggtcaagg
     9421 ttctggacca gttgcgtgag cgcatacgct acttgcatta cagtttacga accgaacagg
     9481 cttatgtcaa ctgggttcgt gcgagctcat cgctagcgcc ttcttgacga gttcttctga
     9541 ttaattaaac tagtgagctg ttgacaatta atcatcgaat ctagtttaat gtgtggaatt
     9601 gtgagcggat aacaattaag cgttaggagg aaacg
//
