LOCUS       sequence-151384-        4558 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template plasmid which encodes kanamycin resistance gene for 
            positive selection and toxin gene (mqsR) 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 4558)
  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 4558)
  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-241
FEATURES             Location/Qualifiers
     source          1..4558
                     /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      2436..2682
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     protein_bind    complement(2735..2768)
                     /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      2817..3173
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(join(3270..4061,4062..4130))
                     /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     3893..3912
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4131..4235)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      4326..4412
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4504..4531
                     /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 tggaaaaacg cacaccacat
     2101 acacgtttga gtcaggttaa aaaacttgtc aatgccgggc aagttcgtac aacacgtagt
     2161 gccctgttaa atgcagatga gttaggtttg gattttgatg gtatgtgtaa tgttatcatt
     2221 ggattatcag agagcgactt ttataaaagc atgaccacct actctgatca tactatctgg
     2281 caggatgttt acagacccag gcttgttaca ggccaggttt atcttaaaat tacggtaatt
     2341 catgacgtac tgatcgtctc gtttaaggag aagtaaggat ccgcgaattc tcatgtttga
     2401 cagcttatca ctgatcagtg aattaatggc gatgacgcat cctcacgata atatccgggt
     2461 aggcgcaatc actttcgtct ctactccgtt acaaagcgag gctgggtatt tcccggcctt
     2521 tctgttatcc gaaatccact gaaagcacag cggctggctg aggagataaa taataaacga
     2581 ggggctgtat gcacaaagca tcttctgttg agttaagaac gagtatcgag atggcacata
     2641 gccttgctca aattggaatc aggtttgtgc caataccagt agaaacagac gaagaagcta
     2701 gctttgcact ggatggggac gtcaggatcg ctctgaagtt cctatacttt ctagagaata
     2761 ggaacttcgg aataggaact aaggaggata ttcatatgga ccatggctaa ttcccatgtc
     2821 agccgttaag tgttcctgtg tcactcaaaa ttgctttgag aggctctaag ggcttctcag
     2881 tgcgttacat ccctggcttg ttgtccacaa ccgttaaacc ttaaaagctt taaaagcctt
     2941 atatattctt ttttttctta taaaacttaa aaccttagag gctatttaag ttgctgattt
     3001 atattaattt tattgttcaa acatgagagc ttagtacgtg aaacatgaga gcttagtacg
     3061 ttagccatga gagcttagta cgttagccat gagggtttag ttcgttaaac atgagagctt
     3121 agtacgttaa acatgagagc ttagtacgtg aaacatgaga gcttagtacg tactatcaac
     3181 aggttgaact gcggatcttg cggccgcaaa aattaaaaat gaagttttaa atcaatctaa
     3241 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     3301 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     3361 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     3421 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     3481 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     3541 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     3601 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     3661 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     3721 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     3781 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     3841 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     3901 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     3961 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     4021 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     4081 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     4141 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     4201 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     4261 gcatcgatgg ccccccgatg gtagtgtggg gtctccccat gcgagagtag ggaactgcca
     4321 ggcatcaaat aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt
     4381 tgtcggtgaa cgctctcctg agtaggacaa atccgccggg agcggatttg aacgttgcga
     4441 agcaacggcc cggagggtgg cgggcaggac gcccgccata aactgccagg catcaaatta
     4501 agcagaaggc catcctgacg gatggccttt ttgcgtggcc agtgccaagc ttgcatgc
//
