LOCUS       sequence-224239-        8035 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Construction of unmarked gene deletions. Broad host range 
            replicative plasmid expressing the I-SceI homing endonuclease .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8035)
  AUTHORS   Flannagan RS, Linn T, Valvano MA
  TITLE     A system for the construction of targeted unmarked gene deletions in
            the genus Burkholderia.
  JOURNAL   Environ Microbiol. 2008 Jun;10(6):1652-60. Epub 2008 Mar 13.
  PUBMED    18341581
REFERENCE   2  (bases 1 to 8035)
  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: "Environ 
            Microbiol."; date: "2008-06"; volume: "10(6)"; pages: "1652-60. Epub
            2008 Mar 13"
COMMENT     Sequence Label: pDAI-SceI
FEATURES             Location/Qualifiers
     source          1..8035
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             721..735
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     primer_bind     complement(793..810)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      963..1049
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1141..1168
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     1936..1955
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2022..2124)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2241..2259)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      3517..4288
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
     CDS             4289..4951
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQRRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     CDS             complement(5479..6678)
                     /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             6784..7434
                     /codon_start=1
                     /gene="tetR"
                     /product="tetracycline resistance regulatory protein"
                     /label=TetR
                     /translation="MTKLQPNTVIRAALDLLNEVGVDGLTTRKLAERLGVQQPALYWHF
                     RNKRALLDALAEAMLAENHTHSVPRADDDWRSFLIGNARSFRQALLAYRDGARIHAGTR
                     PGAPQMETADAQLRFLCEAGFSAGDAVNALMTISYFTVGAVLEEQAGDSDAGERGGTVE
                     QAPLSPLLRAAIDAFDEAGPDAAFEQGLAVIVDGLAKRRLVVRNVEGPRKGDD"
     primer_bind     7535..7554
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        join(7673..7678,7679..7695,7696..7701)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
ORIGIN
        1 atgcatcaaa aaaaccaggt aatgaacctg ggtccgaact ctaaactgct gaaagaatac
       61 aaatcccagc tgatcgaact gaacatcgaa cagttcgaag caggtatcgg tctgatcctg
      121 ggtgatgctt acatccgttc tcgtgatgaa ggtaaaacct actgtatgca gttcgagtgg
      181 aaaaacaaag catacatgga ccacgtatgt ctgctgtacg atcagtgggt actgtccccg
      241 ccgcacaaaa aagaacgtgt taaccacctg ggtaacctgg taatcacctg gggcgcccag
      301 actttcaaac accaagcttt caacaaactg gctaacctgt tcatcgttaa caacaaaaaa
      361 accatcccga acaacctggt tgaaaactac ctgaccccga tgtctctggc atactggttc
      421 atggatgatg gtggtaaatg ggattacaac aaaaactcta ccaacaaatc gatcgtactg
      481 aacacccagt ctttcacttt cgaagaagta gaatacctgg ttaagggtct gcgtaacaaa
      541 ttccaactga actgttacgt aaaaatcaac aaaaacaaac cgatcatcta catcgattct
      601 atgtcttacc tgatcttcta caacctgatc aaaccgtacc tgatcccgca gatgatgtac
      661 aaactgccga acactatctc ctccgaaact ttcctgaaat aatctagaga ctacaaggac
      721 gacgacgacg acaagtgacg acctgcaggc atgcaagctt ggctgttttg gcggatgaga
      781 gaagattttc agcctgatac agattaaatc agaacgcaga agcggtctga taaaacagaa
      841 tttgcctggc ggcagtagcg cggtggtccc acctgacccc atgccgaact cagaagtgaa
      901 acgccgtagc gccgatggta gtgtggggtc tccccatgcg agagtaggga actgccaggc
      961 atcaaataaa acgaaaggct cagtcgaaag actgggcctt tcgttttatc tgttgtttgt
     1021 cggtgaacgc tctcctgagt aggacaaatc cgccgggagc ggatttgaac gctgcgaagc
     1081 aacggcccgg agggtggcgg gcaggacgcc cgccataaac tgccaggcat caaattaagc
     1141 agaaggccat cctgacggat ggcctttttg cgtttctaca aactcttcca ctcactacac
     1201 agagccattt aaacaacatc ccctccccct ttccaccgcg tcagacgccc gtagcagccc
     1261 gctacgggct ttttcatgcc ctgccctagc gtccaagcct cacggccgcg ctcggcctct
     1321 ctggcggcct tctggcgctg aggtctgcct cgtgaagaag gtgttgctga ctcataccag
     1381 gcctgaatcg ccccatcatc cagccagaaa gtgagggagc cacggttgat gagagctttg
     1441 ttgtaggtgg accagttggt gattttgaac ttttgctttg ccacggaacg gtctgcgttg
     1501 tcgggaagat gcgtgatctg atccttcaac tcagcaaaag ttcgatttat tcaacaaagc
     1561 cgccgtcccg tcaagtcagc gtaatgctct gccagtgtta caaccaatta accaattctg
     1621 attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     1681 taccgtattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     1741 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     1801 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     1861 ctgaatccgg tgagaatggc aaaagctaaa aaggccgtaa tatccagctg aacggtctgg
     1921 ttataggtac attgagcaac tgactgaaat gcctcaaaat gttctttacg atgccattgg
     1981 gatatatcaa cggtggtata tccagtgatt tttttctcca ttttagcttc cttagctcct
     2041 gaaaatctcg ataactcaaa aaatacgccc ggtagtgatc ttatttcatt atggtgaaag
     2101 ttggaacctc ttacgtgccg atcaacgtct cattttcgcc aaaagttggc ccagggcttc
     2161 ccggtatcaa cagggacacc aggatttatt tattctgcga agtgatcttc cgtcacaggt
     2221 atttattcga agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     2281 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg cccgcgttcc
     2341 tgctggcgct gggcctgttt ctggcgctgg acttcccgct gttccgtcag cagcttttcg
     2401 cccacggcct tgatgatcgc ggcggccttg gcctgcatat cccgattcaa cggccccagg
     2461 gcgtccagaa cgggcttcag gcgctcccga aggtctcggg ccgtctcttg ggcttgatcg
     2521 gccttcttgc gcatctcacg cgctcctgcg gcggcctgta gggcaggctc atacccctgc
     2581 cgaaccgctt ttgtcagccg gtcggccacg gcttccggcg tctcaacgcg ctttgagatt
     2641 cccagctttt cggccaatcc ctgcggtgca taggcgcgtg gctcgaccgc ttgcgggctg
     2701 atggtgacgt ggcccactgg tggccgctcc agggcctcgt agaacgcctg aatgcgcgtg
     2761 tgacgtgcct tgctgccctc gatgccccgt tgcagcccta gatcggccac agcggccgca
     2821 aacgtggtct ggtcgcgggt catctgcgct ttgttgccga tgaactcctt ggccgacagc
     2881 ctgccgtcct gcgtcagcgg caccacgaac gcggtcatgt gcgggctggt ttcgtcacgg
     2941 tggatgctgg ccgtcacgat gcgatccgcc ccgtacttgt ccgccagcca cttgtgcgcc
     3001 ttctcgaaga acgccgcctg ctgttcttgg ctggccgact tccaccattc cgggctggcc
     3061 gtcatgacgt actcgaccgc caacacagcg tccttgcgcc gcttctctgg cagcaactcg
     3121 cgcagtcggc ccatcgcttc atcggtgctg ctggccgccc agtgctcgtt ctctggcgtc
     3181 ctgctggcgt cagcgttggg cgtctcgcgc tcgcggtagg cgtgcttgag actggccgcc
     3241 acgttgccca ttttcgccag cttcttgcat cgcatgatcg cgtatgccgc catgcctgcc
     3301 cctccctttt ggtgtccaac cggctcgacg ggggcagcgc aaggcggtgc ctccggcggg
     3361 ccactcaatg cttgagtata ctcactagac tttgcttcgc aaagtcgtga ccgcctacgg
     3421 cggctgcggc gccctacggg cttgctctcc gggcttcgcc ctgcgcggtc gctgcgctcc
     3481 cttgccagcc cgtggatatg tggacgatgg ccgcgagcgg ccaccggctg gctcgcttcg
     3541 ctcggcccgt ggacaaccct gctggacaag ctgatggaca ggctgcgcct gcccacgagc
     3601 ttgaccacag ggattgccca ccggctaccc agccttcgac cacataccca ccggctccaa
     3661 ctgcgcggcc tgcggccttg ccccatcaat ttttttaatt ttctctgggg aaaagcctcc
     3721 ggcctgcggc ctgcgcgctt cgcttgccgg ttggacacca agtggaaggc gggtcaaggc
     3781 tcgcgcagcg accgcgcagc ggcttggcct tgacgcgcct ggaacgaccc aagcctatgc
     3841 gagtgggggc agtcgaaggg cgaagcccgc ccgcctgccc cccgagcctc acggcggcga
     3901 gtgcgggggt tccaaggggg cagcgccacc ttgggcaagg ccgaaggccg cgcagtcgat
     3961 caacaagccc cggaggggcc actttttgcc ggagggggag ccgcgccgaa ggcgtggggg
     4021 aaccccgcag gggtgccctt ctttgggcac caaagaacta gatatagggc gaaatgcgaa
     4081 agacttaaaa atcaacaact taaaaaaggg gggtacgcaa cagctcattg cggcaccccc
     4141 cgcaatagct cattgcgtag gttaaagaaa atctgtaatt gactgccact tttacgcaac
     4201 gcataattgt tgtcgcgctg ccgaaaagtt gcagctgatt gcgcatggtg ccgcaaccgt
     4261 gcggcacccc taccgcatgg agataagcat ggccacgcag tccagagaaa tcggcattca
     4321 agccaagaac aagcccggtc actgggtgca aacggaacgc aaagcgcatg aggcgtgggc
     4381 cgggcttatt gcgaggaaac ccacggcggc aatgctgctg catcacctcg tggcgcagat
     4441 gggccaccag aacgccgtgg tggtcagcca gaagacactt tccaagctca tcggacgttc
     4501 tttgcggacg gtccaatacg cagtcaagga cttggtggcc gagcgctgga tctccgtcgt
     4561 gaagctcaac ggccccggca ccgtgtcggc ctacgtggtc aatgaccgcg tggcgtgggg
     4621 ccagccccgc gaccagttgc gcctgtcggt gttcagtgcc gccgtggtgg ttgatcacga
     4681 cgaccaggac gaatcgctgt tggggcatgg cgacctgcgc cgcatcccga ccctgtatcc
     4741 gggcgagcag caactaccga ccggccccgg cgaggagccg cccagccagc ccggcattcc
     4801 gggcatggaa ccagacctgc cagccttgac cgaaacggag gaatgggaac ggcgcgggca
     4861 gcggcgcctg ccgatgcccg atgagccgtg ttttctggac gatggcgagc cgttggagcc
     4921 gccgacacgg gtcacgctgc cgcgccggta gcacttgggt tgcgcagcaa cccgtaagtg
     4981 cgctgttcca gactatcggc tgtagccgcc tcgccgccct ataccttgtc tgcctccccg
     5041 cgttgcgtcg cggtgcatgg agccgggcca cctcgacctg aatggaagcc ggcggcacct
     5101 cgctaacgga ttcaccgttt ttatcaggct ctgggaggca gaataaatga tcatatcgtc
     5161 aattattacc tccacgggga gagcctgagc aaactggcct caggcatttg agaagcacac
     5221 ggtcacactg cttccggtag tcaataaacc ggtaaaccag caatagacat aagcggctat
     5281 ttaacgaccc tgccctgaac cgacgaccgg gtcgaatttg ctttcgaatt tctgccattc
     5341 atccgcttat tatcacttat tcaggcgtag caccaggcgt ttaagtcgac caataaccta
     5401 cggggtctga cgctcagtgg aacgaaatcg atgcgaagaa gttgtccata ttggccacgt
     5461 ttcctatcgt ttccacgatc agcgatcggc tcgttgccct gcgccgctcc aaagcccgcg
     5521 acgcagcgcc ggcaggcaga gcaagtagag ggcagcgcct gcaatccatg cccacccgtt
     5581 ccacgttgtt atagaagccg catagatcgc cgtgaagagg aggggtccga cgatcgaggt
     5641 caggctggtg agcgccgcca gtgagccttg cagctgcccc tgacgttcct catccacctg
     5701 cctggacaac attgcttgca gcgccggcat tccgatgcca cccgaagcaa gcaggaccat
     5761 gatcgggaac gccatccatc cccgtgtcgc gaaggcaagc aggatgtagc ctgtgccgtc
     5821 ggcaatcatt ccgagcatga gtgcccgcct ttcgccgagc cgggcggcta cagggccggt
     5881 gatcattgcc tgggcgagtg aatgcagaat gccaaatgcg gcaagcgaaa tgccgatcgt
     5941 ggtcgcgtcc cagtgaaagc gatcctcgcc gaaaatgacc caaagcgcgg ccggcacctg
     6001 tccgacaagt tgcatgatga agaagaccgc catcagggcg gcgacgacgg tcatgccccg
     6061 ggcccaccgg aacgaagcga gcgggttgag agcctcccgg cgtaacggcc ggcgttcgcc
     6121 tttgtgcgac tccggcaaaa ggaaacagcc cgtcaggaaa ttgaggccgt tcaaggctgc
     6181 cgcggcgaag aacggagcgt ggggggagaa accgcccatc agcccaccga gcacaggtcc
     6241 cgcgaccatc ccgaacccga aacaggcgct catgaagccg aagtgccgcg cgcgctcatc
     6301 gccatcagtg atatcggcaa tataagcgcc ggctaccgcc ccagtcgccc cggtgatgcc
     6361 ggccacgatc cgcccgatat agagaaccca aaggaaaggc gctgtcgcca tgatggcgta
     6421 gtcgacagtg gcgccggcca gcgagacgag caagattggc cgccgcccga aacgatccga
     6481 cagcgcgccc agcacaggtg cgcaggcaaa ttgcaccaac gcatacagcg ccagcagaat
     6541 gccatagtgg gcggtgacgt cgttcgagtg aaccagatcg cgcaggaggc ccggcagcac
     6601 cggcataatc aggccgatgc cgacagcgtc gagcgcgaca gtgctcagaa ttacgatcag
     6661 gggtatgttg ggtttcacgt ctggcctccg gaccagcctc cgctggtccg attgaacgcg
     6721 cggattcttt atcactgata agttggtgga catattatgt ttatcagtga taaagtgtca
     6781 agcatgacaa agttgcagcc gaatacagtg atccgtgccg ccctggacct gttgaacgag
     6841 gtcggcgtag acggtctgac gacacgcaaa ctggcggaac ggttgggggt tcagcagccg
     6901 gcgctttact ggcacttcag gaacaagcgg gcgctgctcg acgcactggc cgaagccatg
     6961 ctggcggaga atcatacgca ttcggtgccg agagccgacg acgactggcg ctcatttctg
     7021 atcgggaatg cccgcagctt caggcaggcg ctgctcgcct accgcgatgg cgcgcgcatc
     7081 catgccggca cgcgaccggg cgcaccgcag atggaaacgg ccgacgcgca gcttcgcttc
     7141 ctctgcgagg cgggtttttc ggccggggac gccgtcaatg cgctgatgac aatcagctac
     7201 ttcactgttg gggccgtgct tgaggagcag gccggcgaca gcgatgccgg cgagcgcggc
     7261 ggcaccgttg aacaggctcc gctctcgccg ctgttgcggg ccgcgataga cgccttcgac
     7321 gaagccggtc cggacgcagc gttcgagcag ggactcgcgg tgattgtcga tggattggcg
     7381 aaaaggaggc tcgttgtcag gaacgttgaa ggaccgagaa agggtgacga ttgatcagga
     7441 ccgctgccgg agcgcaaccc actcactaca gcagagccat cgatcgatct gcacttgaac
     7501 gtgtggccta agcgatatct taggatctcc catcggtgat gtcggcgata taggcgccag
     7561 caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag aggatctgct
     7621 catgtttgac agcttactcg agtatgctag cgatgagctc gcacgaaccc agttgacata
     7681 agcctgttcg gttcgtaaac tgtaatgcaa gtagcgtatg cgctcacgca actggtccag
     7741 aaccttgacc gaacgcagcg gtggtaacgg cgcagtggcg gttttcatgg cttgttatga
     7801 ctgttttttt gtacagtcta tgcctcgggc atccaagcag caagcgcgtt acgccgtggg
     7861 tcgatgtttg atgttatgga gcagcaacga tgttacgcag cagggcagtc gccctaaaac
     7921 aaagttaggc agccgttgtg ctggtgcttt ctgatagttg ttgtggggta ggcagtcaga
     7981 gctcgatttg cttgtcgcca taatagattc acaagaagga ttcggaattc tacat
//
