LOCUS       sequence-201157-        4226 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  LI Gateway cassette.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4226)
  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 4226)
  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: LI C-D Dest
FEATURES             Location/Qualifiers
     source          1..4226
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    89..213
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(250..255,256..273,274..280)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             334..993
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(400..419)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1335..1640
                     /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     1519..1538
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1681..1805)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1879..1895)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1879..1895)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1892..1914)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1903..1919
                     /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(1927..1933,1934..1951,1952..1957))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1972..1993
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2110..2127)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2281..2869)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2361..2380)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(2984..2989,2990..3006,3007..3012)
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     CDS             3201..3734
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
     primer_bind     3821..3839
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3877..3899)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3999..4018
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     join(4212..4226,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(4226,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtgaa ttcgagctcg gtacctcgcg aatgcatcta gatatcggat
       61 cccttgaaga ctgtacgggt ctcacaccac aagtttgtac aaaaaagctg aacgagaaac
      121 gtaaaatgat ataaatatca atatattaaa ttagattttg cataaaaaac agactacata
      181 atactgtaaa acacaacata tccagtcact atggcggccc ctcgaggggc cgcattaggc
      241 accccaggct ttacacttta tgcttccggc tcgtataatg tgtggatttt gagttaggat
      301 ccgtcgagat tttcaggagc taaggaagct aaaatggaga aaaaaatcac tggatatacc
      361 accgttgata tatcccaatg gcatcgtaaa gaacattttg aggcatttca gtcagttgct
      421 caatgtacct ataaccagac cgttcagctg gatattacgg cctttttaaa gaccgtaaag
      481 aaaaataagc acaagtttta tccggccttt attcacattc ttgcccgcct gatgaatgct
      541 catccggaat tccgtatggc aatgaaagac ggtgagctgg tgatatggga tagtgttcac
      601 ccttgttaca ccgttttcca tgagcaaact gaaacgtttt catcgctctg gagtgaatac
      661 cacgacgatt tccggcagtt tctacacata tattcgcaag atgtggcgtg ttacggtgaa
      721 aacctggcct atttccctaa agggtttatt gagaatatgt ttttcgtctc agccaatccc
      781 tgggtgagtt tcaccagttt tgatttaaac gtggccaata tggacaactt cttcgccccc
      841 gttttcacca tgggcaaata ttatacgcaa ggcgacaagg tgctgatgcc gctggcgatt
      901 caggttcatc atgccgtttg tgatggcttc catgtcggca gaatgcttaa tgaattacaa
      961 cagtactgcg atgagtggca gggcggggcg taaagatctg gatccggctt actaaaagcc
     1021 agataacagt atgcgtattt gcgcgctgat ttttgcggta taagaatata tactgatatg
     1081 tatacccgaa gtatgtcaaa aagaggtatg ctatgaagca gcgtattaca gtgacagttg
     1141 acagcgacag ctatcagttg ctcaaggcat atatgatgtc aatatctccg gtctggtaag
     1201 cacaaccatg cagaatgaag cccgtcgtct gcgtgccgaa cgctggaaag cggaaaatca
     1261 ggaagggatg gctgaggtcg cccggtttat tgaaatgaac ggctcttttg ctgacgagaa
     1321 caggggctgg tgaaatgcag tttaaggttt acacctataa aagagagagc cgttatcgtc
     1381 tgtttgtgga tgtacagagt gatattattg acacgcccgg gcgacggatg gtgatccccc
     1441 tggccagtgc acgtctgctg tcagataaag tctcccgtga actttacccg gtggtgcata
     1501 tcggggatga aagctggcgc atgatgacca ccgatatggc cagtgtgccg gtgtccgtta
     1561 tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc
     1621 tgatgttctg gggaatataa atgtcaggct cccttataca cagccagtct gcaggtcgac
     1681 catagtgact ggatatgttg tgttttacag tattatgtag tctgtttttt atgcaaaatc
     1741 taatttaata tattgatatt tatatcattt tacgtttctc gttcagcttt cttgtacaaa
     1801 gtggtgaagg tgagacctct gacgtcttct tgggcccgtc gactgcagag gcctgcatgc
     1861 aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     1921 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     1981 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     2041 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     2101 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     2161 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     2221 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     2281 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     2341 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     2401 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     2461 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     2521 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     2581 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     2641 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     2701 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     2761 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     2821 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     2881 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     2941 catgaggcgc acaccgtgga aacggatgaa ggcacgaacc cagttgacat aagcctgttc
     3001 ggttcgtaaa ctgtaatgca agtagcgtat gcgctcacgc aactggtcca gaaccttgac
     3061 cgaacgcagc ggtggtaacg gcgcagtggc ggttttcatg gcttgttatg actgtttttt
     3121 tgtacagtct atgcctcggg catccaagca gcaagcgcgt tacgccgtgg gtcgatgttt
     3181 gatgttatgg agcagcaacg atgttacgca gcagcaacga tgttacgcag cagggcagtc
     3241 gccctaaaac aaagttaggt ggctcaagta tgggcatcat tcgcacatgt aggctcggcc
     3301 ctgaccaagt caaatccatg cgggctgctc ttgatctttt cggtcgtgag ttcggcgacg
     3361 tagccaccta ctcccaacat cagccggact ccgattacct cgggaacttg ctccgtagta
     3421 agacattcat cgcgcttgct gccttcgacc aagaagcggt tgttggcgct ctcgcggctt
     3481 acgttctgcc caagtttgag cagccgcgta gtgagatcta tatctatgat ctcgcagtct
     3541 ccggagagca ccggaggcag ggcattgcca ccgcgctcat caatctcctc aagcatgagg
     3601 ccaacgcgct tggtgcttat gtgatctacg tgcaagcaga ttacggtgac gatcccgcag
     3661 tggctctcta tacaaagttg ggcatacggg aagaagtgat gcactttgat atcgacccaa
     3721 gtaccgccac ctaacaattc gttcaagccg agatcggctt cccggccgcg gagttgttcg
     3781 gtaaattgtc acaacgccgc tcatgacatt aacctataaa aataggcgta tcacgaggcc
     3841 cttttgtctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga
     3901 gaaggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc
     3961 agcgggtgtt ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact
     4021 gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat
     4081 caggcgccat tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc
     4141 ttcgctatta cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac
     4201 gccagggttt tcccagtcac gacgtt
//
