LOCUS       sequence-198176-        3955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LII backbone vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3955)
  AUTHORS   Wu D, Schandry N, Lahaye T
  TITLE     A modular toolbox for Golden-Gate-based plasmid assembly streamlines
            generation of Ralstonia solanacearum species complex knockout 
            strains and multi-cassette complementation constructs.
  JOURNAL   Mol Plant Pathol. 2017 Oct 27. doi: 10.1111/mpp.12632.
  PUBMED    29077245
REFERENCE   2  (bases 1 to 3955)
  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: "Mol Plant 
            Pathol. 2017 Oct 27. doi: 10.1111/mpp.12632."
COMMENT     Sequence Label: LII destination
FEATURES             Location/Qualifiers
     source          1..3955
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        join(194..199,200..217,218..224)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             278..937
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(344..363)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1279..1584
                     /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     1463..1482
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(1734..1750)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1734..1750)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1747..1769)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1758..1774
                     /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)."
     promoter        complement(join(1782..1788,1789..1806,1807..1812))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1827..1848
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1965..1982)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2136..2724)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2216..2235)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2895..3686,3687..3755))
                     /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     3518..3537
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3756..3860)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3928..3946
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaaccacc tgagaccgcg gcccctcgag gggtcgcatt
      181 aggcacccca ggctttacac tttatgcttc cggctcgtat aatgtgtgga ttttgagtta
      241 ggatccgtcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata
      301 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
      361 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt
      421 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa
      481 tgctcatccg gaattccgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt
      541 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga
      601 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg
      661 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
      721 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc
      781 ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc
      841 gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt
      901 acaacagtac tgcgatgagt ggcagggcgg ggcgtaaaga tctggatccg gcttactaaa
      961 agccagataa cagtatgcgt atttgcgcgc tgatttttgc ggtataagaa tatatactga
     1021 tatgtatacc cgaagtatgt caaaaagagg tatgctatga agcagcgtat tacagtgaca
     1081 gttgacagcg acagctatca gttgctcaag gcatatatga tgtcaatatc tccggtctgg
     1141 taagcacaac catgcagaat gaagcccgtc gtctgcgtgc cgaacgctgg aaagcggaaa
     1201 atcaggaagg gatggctgag gtcgcccggt ttattgaaat gaacggctct tttgctgacg
     1261 agaacagggg ctggtgaaat gcagtttaag gtttacacct ataaaagaga gagccgttat
     1321 cgtctgtttg tggatgtaca gagtgatatt attgacacgc ccgggcgacg gatggtgatc
     1381 cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg
     1441 catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc
     1501 gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt
     1561 aacctgatgt tctggggaat ataaatgtca ggctccctta tacacagcca gtctgcaggt
     1621 cgaccggtct caaagggtgt gaattcgagc tcggtacctc gcgaatgcat ctagatatcg
     1681 gatcccgggc ccgtcgactg cagaggcctg catgcaagct tggcgtaatc atggtcatag
     1741 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     1801 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     1861 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     1921 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     1981 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     2041 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     2101 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     2161 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     2221 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     2281 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     2341 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     2401 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     2461 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     2521 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     2581 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     2641 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     2701 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     2761 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     2821 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     2881 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     2941 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     3001 ttaccatctg gccccagtgc tgcaatgata ccgcgagatc cacgctcacc ggctccagat
     3061 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     3121 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     3181 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     3241 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     3301 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     3361 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     3421 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     3481 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     3541 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     3601 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     3661 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     3721 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     3781 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     3841 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     3901 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtc
//
