LOCUS       sequence-151344-        4549 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template plasmid which encodes a toxin gene (relE) under the control
            of rhamnose induceable promoter (PrhaB) to be used as a negative 
            selection marker..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4549)
  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 4549)
  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-217
FEATURES             Location/Qualifiers
     source          1..4549
                     /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      2427..2673
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     protein_bind    complement(2726..2759)
                     /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      2808..3164
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(join(3261..4052,4053..4121))
                     /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     3884..3903
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4122..4226)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      4317..4403
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4495..4522
                     /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 tggcgtattt tctggatttt
     2101 gacgagcggg cactaaagga atggcgaaag ctgggctcga cggtacgtga acagttgaaa
     2161 aagaagctgg ttgaagtact tgagtcaccc cggattgaag caaacaagct ccgtggtatg
     2221 cctgattgtt acaagattaa gctccggtct tcaggctatc gccttgtata ccaggttata
     2281 gacgagaaag ttgtcgtttt cgtgatttct gttgggaaaa gagaacgctc ggaagtatat
     2341 agcgaggcgg tcaaacgcat tctctgagga tccgcgaatt ctcatgtttg acagcttatc
     2401 actgatcagt gaattaatgg cgatgacgca tcctcacgat aatatccggg taggcgcaat
     2461 cactttcgtc tctactccgt tacaaagcga ggctgggtat ttcccggcct ttctgttatc
     2521 cgaaatccac tgaaagcaca gcggctggct gaggagataa ataataaacg aggggctgta
     2581 tgcacaaagc atcttctgtt gagttaagaa cgagtatcga gatggcacat agccttgctc
     2641 aaattggaat caggtttgtg ccaataccag tagaaacaga cgaagaagct agctttgcac
     2701 tggatgggga cgtcaggatc gctctgaagt tcctatactt tctagagaat aggaacttcg
     2761 gaataggaac taaggaggat attcatatgg accatggcta attcccatgt cagccgttaa
     2821 gtgttcctgt gtcactcaaa attgctttga gaggctctaa gggcttctca gtgcgttaca
     2881 tccctggctt gttgtccaca accgttaaac cttaaaagct ttaaaagcct tatatattct
     2941 tttttttctt ataaaactta aaaccttaga ggctatttaa gttgctgatt tatattaatt
     3001 ttattgttca aacatgagag cttagtacgt gaaacatgag agcttagtac gttagccatg
     3061 agagcttagt acgttagcca tgagggttta gttcgttaaa catgagagct tagtacgtta
     3121 aacatgagag cttagtacgt gaaacatgag agcttagtac gtactatcaa caggttgaac
     3181 tgcggatctt gcggccgcaa aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     3241 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     3301 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     3361 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     3421 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     3481 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     3541 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     3601 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     3661 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     3721 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     3781 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     3841 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     3901 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     3961 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     4021 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     4081 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     4141 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     4201 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgcatcgatg
     4261 gccccccgat ggtagtgtgg ggtctcccca tgcgagagta gggaactgcc aggcatcaaa
     4321 taaaacgaaa ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga
     4381 acgctctcct gagtaggaca aatccgccgg gagcggattt gaacgttgcg aagcaacggc
     4441 ccggagggtg gcgggcagga cgcccgccat aaactgccag gcatcaaatt aagcagaagg
     4501 ccatcctgac ggatggcctt tttgcgtggc cagtgccaag cttgcatgc
//
