LOCUS       sequence-223651-        3956 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 3956)
  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 3956)
  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..3956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        join(195..200,201..218,219..225)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             279..938
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(345..364)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1280..1585
                     /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     1464..1483
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(1735..1751)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1735..1751)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1748..1770)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1759..1775
                     /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(1783..1789,1790..1807,1808..1813))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1828..1849
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1966..1983)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2137..2725)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2217..2236)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2896..3687,3688..3756))
                     /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     3519..3538
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3757..3861)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3929..3947
                     /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 cttaactcac ctgagaccgc ggcccctcga ggggtcgcat
      181 taggcacccc aggctttaca ctttatgctt ccggctcgta taatgtgtgg attttgagtt
      241 aggatccgtc gagattttca ggagctaagg aagctaaaat ggagaaaaaa atcactggat
      301 ataccaccgt tgatatatcc caatggcatc gtaaagaaca ttttgaggca tttcagtcag
      361 ttgctcaatg tacctataac cagaccgttc agctggatat tacggccttt ttaaagaccg
      421 taaagaaaaa taagcacaag ttttatccgg cctttattca cattcttgcc cgcctgatga
      481 atgctcatcc ggaattccgt atggcaatga aagacggtga gctggtgata tgggatagtg
      541 ttcacccttg ttacaccgtt ttccatgagc aaactgaaac gttttcatcg ctctggagtg
      601 aataccacga cgatttccgg cagtttctac acatatattc gcaagatgtg gcgtgttacg
      661 gtgaaaacct ggcctatttc cctaaagggt ttattgagaa tatgtttttc gtctcagcca
      721 atccctgggt gagtttcacc agttttgatt taaacgtggc caatatggac aacttcttcg
      781 cccccgtttt caccatgggc aaatattata cgcaaggcga caaggtgctg atgccgctgg
      841 cgattcaggt tcatcatgcc gtttgtgatg gcttccatgt cggcagaatg cttaatgaat
      901 tacaacagta ctgcgatgag tggcagggcg gggcgtaaag atctggatcc ggcttactaa
      961 aagccagata acagtatgcg tatttgcgcg ctgatttttg cggtataaga atatatactg
     1021 atatgtatac ccgaagtatg tcaaaaagag gtatgctatg aagcagcgta ttacagtgac
     1081 agttgacagc gacagctatc agttgctcaa ggcatatatg atgtcaatat ctccggtctg
     1141 gtaagcacaa ccatgcagaa tgaagcccgt cgtctgcgtg ccgaacgctg gaaagcggaa
     1201 aatcaggaag ggatggctga ggtcgcccgg tttattgaaa tgaacggctc ttttgctgac
     1261 gagaacaggg gctggtgaaa tgcagtttaa ggtttacacc tataaaagag agagccgtta
     1321 tcgtctgttt gtggatgtac agagtgatat tattgacacg cccgggcgac ggatggtgat
     1381 ccccctggcc agtgcacgtc tgctgtcaga taaagtctcc cgtgaacttt acccggtggt
     1441 gcatatcggg gatgaaagct ggcgcatgat gaccaccgat atggccagtg tgccggtctc
     1501 cgttatcggg gaagaagtgg ctgatctcag ccaccgcgaa aatgacatca aaaacgccat
     1561 taacctgatg ttctggggaa tataaatgtc aggctccctt atacacagcc agtctgcagg
     1621 tcgaccggtc tcaaagggtg tgaattcgag ctcggtacct cgcgaatgca tctagatatc
     1681 ggatcccggg cccgtcgact gcagaggcct gcatgcaagc ttggcgtaat catggtcata
     1741 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     1801 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     1861 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     1921 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     1981 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2041 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2101 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2161 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2221 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     2281 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     2341 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     2401 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     2461 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     2521 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     2581 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     2641 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     2701 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     2761 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     2821 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     2881 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     2941 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3001 cttaccatct ggccccagtg ctgcaatgat accgcgagat ccacgctcac cggctccaga
     3061 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3121 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3181 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3241 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     3301 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     3361 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     3421 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     3481 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     3541 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     3601 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     3661 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     3721 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     3781 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     3841 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac
     3901 cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
