LOCUS       sequence-260209-        7372 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Broad host range vector with toxin-antitoxin stability system for 
            maintenance in the absence of antibiotic selection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7372)
  AUTHORS   Burbank L, Wei W
  TITLE     Broad-Host-Range Plasmids for Constitutive and Inducible Gene 
            Expression in the Absence of Antibiotic Selection.
  JOURNAL   Microbiol Resour Announc. 2019 Sep 5;8(36). pii: 8/36/e00769-19. 
            doi: 10.1128/MRA.00769-19.
  PUBMED    31488530
REFERENCE   2  (bases 1 to 7372)
  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: "Microbiol 
            Resour Announc."; date: "2019-09-5"; volume: "8(36). pii: 
            8/36/e00769-19. doi"; pages: " 10.1128/MRA.00769-19"
COMMENT     Sequence Label: pBBR4pemIK-GW
FEATURES             Location/Qualifiers
     source          1..7372
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(1669..2460,2461..2529))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2292..2311
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2530..2601)
                     /gene="bla"
                     /label=AmpR promoter
     protein_bind    2829..2850
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2865..2870,2871..2888,2889..2895)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2903..2919
                     /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     2908..2930
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2927..2943
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2927..2943
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2962..2982
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2964..2982
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     3011..3027
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     3012..3028
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3044..3168
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3193..3198,3199..3216,3217..3223)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3277..3936
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3343..3362)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4278..4583
                     /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     4462..4481
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4624..4748)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(5480..5496)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5480..5496)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(5528..5547)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5529..5547)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5557..5574)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5557..5573)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5566..5588)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             complement(6282..6944)
                     /codon_start=1
                     /product="replication protein for the broad-host-range 
                     plasmid pBBR1 from Bordetella bronchiseptica"
                     /label=pBBR1 Rep
                     /translation="MATQSREIGIQAKNKPGHWVQTERKAHEAWAGLIARKPTAAMLLH
                     HLVAQMGHQNAVVVSQKTLSKLIGRSLRTVQYAVKDLVAERWISVVKLNGPGTVSAYVV
                     NDRVAWGQPRDQLRLSVFSAAVVVDHDDQDESLLGHGDLRRIPTLYPGEQQLPTGPGEE
                     PPSQPGIPGMEPDLPALTETEEWERRGQQRLPMPDEPCFLDDGEPLEPPTRVTLPRR"
     rep_origin      join(6945..7372,1..344)
                     /label=pBBR1 oriV
                     /note="replication origin of the broad-host-range plasmid 
                     pBBR1 from Bordetella bronchiseptica; requires the pBBR1 
                     Rep protein for replication"
ORIGIN
        1 cccttcgact gcccccactc gcataggctt gggtcgttcc aggcgcgtca aggccaagcc
       61 gctgcgcggt cgctgcgcga gccttgaccc gccttccact tggtgtccaa ccggcaagcg
      121 aagcgcgcag gccgcaggcc ggaggctttt ccccagagaa aattaaaaaa attgatgggg
      181 caaggccgca ggccgcgcag ttggagccgg tgggtatgtg gtcgaaggct gggtagccgg
      241 tgggcaatcc ctgtggtcaa gctcgtgggc aggcgcagcc tgtccatcag cttgtccagc
      301 agggttgtcc acgggccgag cgaagcgagc cagccggtgg ccgctcgcgg ccatcgtcca
      361 catatccacg ggctggcaag ggagcgcagc gaccgcgcag ggcgaagccc ggagagcaag
      421 cccgtagggc gccgcagccg ccgtaggcgg tcacgacttt gcgaagcaaa gtctagtgag
      481 tatactcaag cattgagtgg cccgccggag gcaccgcctt gcgctgcccc cgtcgagccg
      541 gttggacacc aaaagggagg ggcaggcatg gcggcatacg cgatcatgcg atgcaagaag
      601 ctggcgaaaa tgggcaacgt ggcggccagt ctcaagcacg cctaccgcga gcgcgagacg
      661 cccaacgctg acgccagcag gacgccagag aacgagcact gggcggccag cagcaccgat
      721 gaagcgatgg gccgactgcg cgagttgctg ccagagaagc ggcgcaagga cgctgtgttg
      781 gcggtcgagt acgtcatgac ggccagcccg gaatggtgga agtcggccag ccaagaacag
      841 caggcggcgt tcttcgagaa ggcgcacaag tggctggcgg acaagtacgg ggcggatcgc
      901 atcgtgacgg ccagcatcca ccgtgacgaa accagcccgc acatgaccgc gttcgtggtg
      961 ccgctgacgc aggacggcag gctgtcggcc aaggagttca tcggcaacaa agcgcagatg
     1021 acccgcgacc agaccacgtt tgcggccgct gtggccgatc tagggctgca acggggcatc
     1081 gagggcagca aggcacgtca cacgcgcatt caggcgttct acgaggccct ggagcggcca
     1141 ccagtgggcc acgtcaccat cagcccgcaa gcggtcgagc cacgcgccta tgcaccgcag
     1201 ggattggccg aaaagctggg aatctcaaag cgcgttgaga cgccggaagc cgtggccgac
     1261 cggctgacaa aagcggttcg gcaggggtat gagcctgccc tacaggccgc cgcaggagcg
     1321 cgtgagatgc gcaagaaggc cgatcaagcc caagagacgg cccgagacct tcgggagcgc
     1381 ctgaagcccg ttctggacgc cctggggccg ttgaatcggg atatgcaggc caaggccgcc
     1441 gcgatcatca aggccgtggg cgaaaagctg ctgacggaac agcgggaagt ccagcgccag
     1501 aaacaggccc agcgccagca ggaacgcggg cgcgcacatt tccccgaaaa gtgccacctg
     1561 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     1621 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     1681 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     1741 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     1801 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     1861 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     1921 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     1981 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     2041 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     2101 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     2161 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     2221 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     2281 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     2341 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     2401 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     2461 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     2521 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     2581 gagcggatac atatttgaat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg
     2641 ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat
     2701 gaattacaac agtttttatg catgcgccca atacgcaaac cgcctctccc cgcgcgttgg
     2761 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc
     2821 aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca ctttatgctt
     2881 ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg aaacagctat
     2941 gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctgggtacc
     3001 gggccccccc tcgaggtcga cggtatcgat aagcttgata tcaacaagtt tgtacaaaaa
     3061 agctgaacga gaaacgtaaa atgatataaa tatcaatata ttaaattaga ttttgcataa
     3121 aaaacagact acataatact gtaaaacaca acatatccag tcactatggc ggccgcatta
     3181 ggcaccccag gctttacact ttatgcttcc ggctcgtata atgtgtggat tttgagttag
     3241 gatccgtcga gattttcagg agctaaggaa gctaaaatgg agaaaaaaat cactggatat
     3301 accaccgttg atatatccca atggcatcgt aaagaacatt ttgaggcatt tcagtcagtt
     3361 gctcaatgta cctataacca gaccgttcag ctggatatta cggccttttt aaagaccgta
     3421 aagaaaaata agcacaagtt ttatccggcc tttattcaca ttcttgcccg cctgatgaat
     3481 gctcatccgg aattccgtat ggcaatgaaa gacggtgagc tggtgatatg ggatagtgtt
     3541 cacccttgtt acaccgtttt ccatgagcaa actgaaacgt tttcatcgct ctggagtgaa
     3601 taccacgacg atttccggca gtttctacac atatattcgc aagatgtggc gtgttacggt
     3661 gaaaacctgg cctatttccc taaagggttt attgagaata tgtttttcgt ctcagccaat
     3721 ccctgggtga gtttcaccag ttttgattta aacgtggcca atatggacaa cttcttcgcc
     3781 cccgttttca ccatgggcaa atattatacg caaggcgaca aggtgctgat gccgctggcg
     3841 attcaggttc atcatgccgt ttgtgatggc ttccatgtcg gcagaatgct taatgaatta
     3901 caacagtact gcgatgagtg gcagggcggg gcgtaaagat ctggatccgg cttactaaaa
     3961 gccagataac agtatgcgta tttgcgcgct gatttttgcg gtataagaat atatactgat
     4021 atgtataccc gaagtatgtc aaaaagaggt atgctatgaa gcagcgtatt acagtgacag
     4081 ttgacagcga cagctatcag ttgctcaagg catatatgat gtcaatatct ccggtctggt
     4141 aagcacaacc atgcagaatg aagcccgtcg tctgcgtgcc gaacgctgga aagcggaaaa
     4201 tcaggaaggg atggctgagg tcgcccggtt tattgaaatg aacggctctt ttgctgacga
     4261 gaacaggggc tggtgaaatg cagtttaagg tttacaccta taaaagagag agccgttatc
     4321 gtctgtttgt ggatgtacag agtgatatta ttgacacgcc cgggcgacgg atggtgatcc
     4381 ccctggccag tgcacgtctg ctgtcagata aagtctcccg tgaactttac ccggtggtgc
     4441 atatcgggga tgaaagctgg cgcatgatga ccaccgatat ggccagtgtg ccggtctccg
     4501 ttatcgggga agaagtggct gatctcagcc accgcgaaaa tgacatcaaa aacgccatta
     4561 acctgatgtt ctggggaata taaatgtcag gctcccttat acacagccag tctgcaggtc
     4621 gaccatagtg actggatatg ttgtgtttta cagtattatg tagtctgttt tttatgcaaa
     4681 atctaattta atatattgat atttatatca ttttacgttt ctcgttcagc tttcttgtac
     4741 aaagtggttg atatcgaatt cgattttcgc tataagcgta taaatggagg aattatatta
     4801 tactttaata taacttaacg aaagaagccc atcatgcaca ccacaaacct tcgtaaagtc
     4861 ggtggatcga tcatgctggc cgttcccccc gctttccttg atcagcttca tcttgaggtc
     4921 ggcgcaacgg tcggcttagc cgtcacagac ggtcgtctgg tcattgagcc agtcctacat
     4981 cctcagtaca cgctggatca actgctggcc gaagcggaag cctccggggc ttacccgttg
     5041 ccgcccgagg aacgtgaatg ggtcgatgct ccagcagtag gacgcgaact gatatgaaac
     5101 gcggcgatgt ttacatggtc gatctggagc caaccgccgg acacgaacaa cgcggtcacc
     5161 gtccggtggt tgttatctcc agtgagcgat tcaatcggct tacctcttgt ccggtgatcc
     5221 ttccaatcac aaacggcggc gagttcgcct cccgcctcgg gttcgctgtg gaattactag
     5281 gtacgattac aacgggcgtc gttcgatgtg atcaaccacg tgcgctcgac ttactcgctc
     5341 gcaatgcccg caaagtggaa agcctaccac caaacatctt ggccgatgtc ttggcaaaag
     5401 ctgtcaccat cttccagtaa tttttggggc aagttcaggc cactgcaacc aatcactagt
     5461 gaattcctgc agcccggggg atccactagt tctagagcgg ccgccaccgc ggtggagctc
     5521 caattcgccc tatagtgagt cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg
     5581 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     5641 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     5701 gaatggcgaa tggaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     5761 taaatcagct cattttttaa ccaataggcc gtactgcgat gagtggcagg gcggggcgta
     5821 atttttttaa ggcagttatt ggtgccctta aacgcctggt tgctacgcct gaataagtga
     5881 taataagcgg atgaatggca gaaattcgaa agcaaattcg acccggtcgt cggttcaggg
     5941 cagggtcgtt aaatagccgc ttatgtctat tgctggttta ccggtttatt gactaccgga
     6001 agcagtgtga ccgtgtgctt ctcaaatgcc tgaggccagt ttgctcaggc tctccccgtg
     6061 gaggtaataa ttgacgatat gatcatttat tctgcctccc agagcctgat aaaaacggtg
     6121 aatccgttag cgaggtgccg ccggcttcca ttcaggtcga ggtggcccgg ctccatgcac
     6181 cgcgacgcaa cgcggggagg cagacaaggt atagggcggc gaggcggcta cagccgatag
     6241 tctggaacag cgcacttacg ggttgctgcg caacccaagt gctaccggcg cggcagcgtg
     6301 acccgtgtcg gcggctccaa cggctcgcca tcgtccagaa aacacggctc atcgggcatc
     6361 ggcaggcgct gctgcccgcg ccgttcccat tcctccgttt cggtcaaggc tggcaggtct
     6421 ggttccatgc ccggaatgcc gggctggctg ggcggctcct cgccggggcc ggtcggtagt
     6481 tgctgctcgc ccggatacag ggtcgggatg cggcgcaggt cgccatgccc caacagcgat
     6541 tcgtcctggt cgtcgtgatc aaccaccacg gcggcactga acaccgacag gcgcaactgg
     6601 tcgcggggct ggccccacgc cacgcggtca ttgaccacgt aggccgacac ggtgccgggg
     6661 ccgttgagct tcacgacgga gatccagcgc tcggccacca agtccttgac tgcgtattgg
     6721 accgtccgca aagaacgtcc gatgagcttg gaaagtgtct tctggctgac caccacggcg
     6781 ttctggtggc ccatctgcgc cacgaggtga tgcagcagca ttgccgccgt gggtttcctc
     6841 gcaataagcc cggcccacgc ctcatgcgct ttgcgttccg tttgcaccca gtgaccgggc
     6901 ttgttcttgg cttgaatgcc gatttctctg gactgcgtgg ccatgcttat ctccatgcgg
     6961 taggggtgcc gcacggttgc ggcaccatgc gcaatcagct gcaacttttc ggcagcgcga
     7021 caacaattat gcgttgcgta aaagtggcag tcaattacag attttcttta acctacgcaa
     7081 tgagctattg cggggggtgc cgcaatgagc tgttgcgtac cccccttttt taagttgttg
     7141 atttttaagt ctttcgcatt tcgccctata tctagttctt tggtgcccaa agaagggcac
     7201 ccctgcgggg ttcccccacg ccttcggcgc ggctccccct ccggcaaaaa gtggcccctc
     7261 cggggcttgt tgatcgactg cgcggccttc ggccttgccc aaggtggcgc tgcccccttg
     7321 gaacccccgc actcgccgcc gtgaggctcg gggggcaggc gggcgggctt cg
//
