LOCUS       sequence-151373-        4612 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Template plasmid which encodes kanamycin resistance gene for 
            positive selection and toxin gene (chpB) under the control of 
            rhamnose induceable promoter (PrhaB) for negative selection..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4612)
  AUTHORS   Khetrapal V, Mehershahi K, Rafee S, Chen S, Lim CL, Chen SL
  TITLE     A set of powerful negative selection systems for unmodified 
            Enterobacteriaceae.
  JOURNAL   Nucleic Acids Res. 2015 Jul 27;43(13):e83. doi: 10.1093/nar/gkv248. 
            Epub 2015 Mar 23.
  PUBMED    25800749
REFERENCE   2  (bases 1 to 4612)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res."; date: "2015-07-27"; volume: "43(13):e83. doi"; pages: "
            10.1093/nar/gkv248. Epub 2015 Mar 23"
COMMENT     Sequence Label: pSLC-238
FEATURES             Location/Qualifiers
     source          1..4612
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(51..98)
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             459..1253
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(513..532)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1123..1142
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     terminator      1530..1616
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      1708..1735
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     regulatory      1789..1798
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        1938..2056
                     /label=rhaB promoter
                     /note="promoter of the E. coli rhaBAD operon, conferring 
                     tight induction with L-rhamnose and repression with 
                     D-glucose in the presence of RhaR and RhaS (Giacalone et 
                     al., 2006)"
     terminator      2490..2736
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     protein_bind    complement(2789..2822)
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     rep_origin      2871..3227
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(join(3324..4115,4116..4184))
                     /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     3947..3966
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4185..4289)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      4380..4466
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4558..4585
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 agattgcagc attacacgtc ttgagcgatt gtgtaggctg gagctgcttc gaagttccta
       61 tactttctag agaataggaa cttcggaata ggaacttcaa gatcccctca cgctgccgca
      121 agcactcagg gcgcaagggc tgctaaagga agcggaacac gtagaaagcc agtccgcaga
      181 aacggtgctg accccggatg aatgtcagct actgggctat ctggacaagg gaaaacgcaa
      241 gcgcaaagag aaagcaggta gcttgcagtg ggcttacatg gcgatagcta gactgggcgg
      301 ttttatggac agcaagcgaa ccggaattgc cagctggggc gccctctggt aaggttggga
      361 agccctgcaa agtaaactgg atggctttct tgccgccaag gatctgatgg cgcaggggat
      421 caagatctga tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc
      481 acgcaggttc tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga
      541 caatcggctg ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt
      601 ttgtcaagac cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat
      661 cgtggctggc cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg
      721 gaagggactg gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg
      781 ctcctgccga gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc
      841 cggctacctg cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga
      901 tggaagccgg tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag
      961 ccgaactgtt cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc
     1021 atggcgatgc ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg
     1081 actgtggccg gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata
     1141 ttgctgaaga gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg
     1201 ctcccgattc gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac
     1261 tctggggttc gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc
     1321 caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat
     1381 gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccagct tcaaaagcga
     1441 tcctccgcgg cccaaagtgc cacctgcatc gatggccccc gatggtagtg tggggtctcc
     1501 ccatgcgaga gtagggaact gccaggcatc aaataaaacg aaaggctcag tcgaaagact
     1561 gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct cctgagtagg acaaatccgc
     1621 cgggagcgga tttgaacgtt gcgaagcaac ggcccggagg gtggcgggca ggacgcccgc
     1681 cataaactgc caggcatcaa attaagcaga aggccatcct gacggatggc ctttttgcgt
     1741 ggccagtgcc aagcttgcat gcctgcagct gtccggaact agtcgaggcc atggtggcct
     1801 cctgatgtcg tcaacacggc gaaatagtaa tcacgaggtc aggttcttac cttaaatttt
     1861 cgacggaaaa ccacgtaaaa aacgtcgatt tttcaagata cagcgtgaat tttcaggaaa
     1921 tgcggtgagc atcacatcac cacaattcag caaattgtga acatcatcac gttcatcttt
     1981 ccctggttgc caatggccca ttttcctgtc agtaacgaga aggtcgcgaa ttcaggcgct
     2041 ttttagactg gtcgtaatga aattcagcag gatcaccata tggtaaagaa aagtgaattt
     2101 gaacggggag acattgtgct ggttggcttt gatccagcaa gcggccatga acagcaaggt
     2161 gctggtcgac ctgcgcttgt gctctccgtt caagccttta atcaactggg aatgacgctg
     2221 gtggccccca ttacgcaggg cggaaatttt gcccgttatg ccggatttag cgttccttta
     2281 cattgcgaag aaggcgatgt gcacggcgtg gtgctggtga atcaggtgcg gatgatggat
     2341 ctacacgccc ggctggcaaa gcgtattggt ctggctgcgg atgaggtggt ggaagaggcg
     2401 ttattacgct tgcaggcggt ggtggaataa ggatccgcga attctcatgt ttgacagctt
     2461 atcactgatc agtgaattaa tggcgatgac gcatcctcac gataatatcc gggtaggcgc
     2521 aatcactttc gtctctactc cgttacaaag cgaggctggg tatttcccgg cctttctgtt
     2581 atccgaaatc cactgaaagc acagcggctg gctgaggaga taaataataa acgaggggct
     2641 gtatgcacaa agcatcttct gttgagttaa gaacgagtat cgagatggca catagccttg
     2701 ctcaaattgg aatcaggttt gtgccaatac cagtagaaac agacgaagaa gctagctttg
     2761 cactggatgg ggacgtcagg atcgctctga agttcctata ctttctagag aataggaact
     2821 tcggaatagg aactaaggag gatattcata tggaccatgg ctaattccca tgtcagccgt
     2881 taagtgttcc tgtgtcactc aaaattgctt tgagaggctc taagggcttc tcagtgcgtt
     2941 acatccctgg cttgttgtcc acaaccgtta aaccttaaaa gctttaaaag ccttatatat
     3001 tctttttttt cttataaaac ttaaaacctt agaggctatt taagttgctg atttatatta
     3061 attttattgt tcaaacatga gagcttagta cgtgaaacat gagagcttag tacgttagcc
     3121 atgagagctt agtacgttag ccatgagggt ttagttcgtt aaacatgaga gcttagtacg
     3181 ttaaacatga gagcttagta cgtgaaacat gagagcttag tacgtactat caacaggttg
     3241 aactgcggat cttgcggccg caaaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3301 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3361 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3421 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     3481 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     3541 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3601 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3661 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3721 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3781 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3841 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3901 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3961 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4021 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4081 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4141 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4201 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4261 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgcatcg
     4321 atggcccccc gatggtagtg tggggtctcc ccatgcgaga gtagggaact gccaggcatc
     4381 aaataaaacg aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg
     4441 tgaacgctct cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gcgaagcaac
     4501 ggcccggagg gtggcgggca ggacgcccgc cataaactgc caggcatcaa attaagcaga
     4561 aggccatcct gacggatggc ctttttgcgt ggccagtgcc aagcttgcat gc
//
