LOCUS       sequence-134708-        4726 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template plasmid which encodes kanamycin resistance gene for 
            positive selection and toxin gene (yhaV) 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 4726)
  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 4726)
  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: "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-239
FEATURES             Location/Qualifiers
     source          1..4726
                     /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      2604..2850
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     protein_bind    complement(2903..2936)
                     /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      2985..3341
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(join(3438..4229,4230..4298))
                     /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     4061..4080
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4299..4403)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      4494..4580
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4672..4699
                     /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 tggattttcc acaaagggtt
     2101 aatggttggg cgctatatgc tcatccctgt tttcaggaaa cctacgacgc tttagttgcc
     2161 gaagtcgaga cattaaaggg aaaagatcct gaaaattatc agagaaaagc cgccacaaag
     2221 ttattggcgg tagtccataa agtgattgag gagcatatca cggtcaatcc atcatcaccg
     2281 gcattccgtc atggcaagtc gttaggctct gggaaaaata aagactggtc acgggtaaaa
     2341 tttggtgctg gtcgttatcg tctcttcttt cgttatagtg aaaaagagaa agtcatcatt
     2401 ctgggatgga tgaacgatga aaacactctg cgcacctacg gtaaaaaaac agatgcctat
     2461 accgtattca gcaaaatgtt aaaaagagga catcctcctg ccgactggga aaccctcacc
     2521 cgagaaacag aagaaaccca ttgaggatcc gcgaattctc atgtttgaca gcttatcact
     2581 gatcagtgaa ttaatggcga tgacgcatcc tcacgataat atccgggtag gcgcaatcac
     2641 tttcgtctct actccgttac aaagcgaggc tgggtatttc ccggcctttc tgttatccga
     2701 aatccactga aagcacagcg gctggctgag gagataaata ataaacgagg ggctgtatgc
     2761 acaaagcatc ttctgttgag ttaagaacga gtatcgagat ggcacatagc cttgctcaaa
     2821 ttggaatcag gtttgtgcca ataccagtag aaacagacga agaagctagc tttgcactgg
     2881 atggggacgt cgatcctgct ctgaagttcc tatactttct agagaatagg aacttcggaa
     2941 taggaactaa ggaggatatt catatggacc atggctaatt cccatgtcag ccgttaagtg
     3001 ttcctgtgtc actgaaaatt gctttgagag gctctaaggg cttctcagtg cgttacatcc
     3061 ctggcttgtt gtccacaacc gttaaacctt aaaagcttta aaagccttat atattctttt
     3121 ttttcttata aaacttaaaa ccttagaggc tatttaagtt gctgatttat attaatttta
     3181 ttgttcaaac atgagagctt agtacgtgaa acatgagagc ttagtacgtt agccatgaga
     3241 gcttagtacg ttagccatga gggtttagtt cgttaaacat gagagcttag tacgttaaac
     3301 atgagagctt agtacgtgaa acatgagagc ttagtacgta ctatcaacag gttgaactgc
     3361 ggatcttgcg gccgcaaaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     3421 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     3481 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     3541 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     3601 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     3661 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     3721 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     3781 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     3841 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     3901 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     3961 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     4021 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     4081 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     4141 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     4201 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     4261 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     4321 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     4381 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctgc atcgatggcc
     4441 ccccgatggt agtgtggggt ctccccatgc gagagtaggg aactgccagg catcaaataa
     4501 aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg
     4561 ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg
     4621 gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca
     4681 tcctgacgga tggccttttt gcgtggccag tgccaagctt gcatgc
//
