LOCUS       sequence-151367-        4755 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Template plasmid which encodes kanamycin resistance gene for 
            positive selection and toxin gene (tse2) 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 4755)
  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 4755)
  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-246
FEATURES             Location/Qualifiers
     source          1..4755
                     /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      complement(1486..1732)
                     /label=lambda tL3 terminator
                     /note="transcription terminator tL3 from phage lambda"
     promoter        complement(2309..2427)
                     /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)"
     regulatory      2567..2576
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     terminator      complement(2630..2657)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(2749..2835)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     protein_bind    complement(2932..2965)
                     /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      3014..3370
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(join(3467..4258,4259..4327))
                     /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     4090..4109
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4328..4432)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      4523..4609
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      4701..4728
                     /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 cccatccagt gcaaagctag cttcttcgtc tgtttctact ggtattggca
     1501 caaacctgat tccaatttga gcaaggctat gtgccatctc gatactcgtt cttaactcaa
     1561 cagaagatgc tttgtgcata cagcccctcg tttattattt atctcctcag ccagccgctg
     1621 tgctttcagt ggatttcgga taacagaaag gccgggaaat acccagcctc gctttgtaac
     1681 ggagtagaga cgaaagtgat tgcgcctacc cggatattat cgtgaggatg cgtcatcgcc
     1741 attaattcac tgatcagtga taagctgtca aacatgagaa ttcgcggatc cctagagccc
     1801 gcgggctttt tcccattggc ggcggatggc gttgcgcacg aagttgtcca gttggcccat
     1861 gaggacctcc cggtacatcg gcttggccgg catgatggtg tagtgggtgg cttgcaggcg
     1921 cttgttgtag ctgtcctgct tcaccttaag gtccggcggg atgtcggtgc cgtcgggtat
     1981 cacgaagtcg ccgtcggcgc ggcgcagcac gccggcgcgg tcgaacaccg aggtgccgcc
     2041 ggtgtccggg gcgtagaggc cgtcgcggcc ggcgacgatg tccggcgcgc gggcggggac
     2101 gccaggggtg ttggggctgt cgaaggtcgg atgcagcagc ggggcgactt ccgcagcctc
     2161 ggcgagttcc ttgtcgggat gcaggtagcg accgacgtcg ccgtcgtagt tggccttgaa
     2221 taccgcccgg tagagggtga ggctggtttt ctcgtagtcg taggacatct attttacctc
     2281 ctcatatggt gatcctgctg aatttcatta cgaccagtct aaaaagcgcc tgaattcgcg
     2341 accttctcgt tactgacagg aaaatgggcc attggcaacc agggaaagat gaacgtgatg
     2401 atgttcacaa tttgctgaat tgtggtgatg tgatgctcac cgcatttcct gaaaattcac
     2461 gctgtatctt gaaaaatcga cgttttttac gtggttttcc gtcgaaaatt taaggtaaga
     2521 acctgacctc gtgattacta tttcgccgtg ttgacgacat caggaggcca ccatggcctc
     2581 gactagttcc ggacagctgc aggcatgcaa gcttggcact ggccacgcaa aaaggccatc
     2641 cgtcaggatg gccttctgct taatttgatg cctggcagtt tatggcgggc gtcctgcccg
     2701 ccaccctccg ggccgttgct tcgcaacgtt caaatccgct cccggcggat ttgtcctact
     2761 caggagagcg ttcaccgaca aacaacagat aaaacgaaag gcccagtctt tcgactgagc
     2821 ctttcgtttt atttgatgcc tggcagttcc ctactctcgc atggggagac cccacactac
     2881 catcgggggc catcgatgca ggtggcactt tggggacgtc aggatcgctc tgaagttcct
     2941 atactttcta gagaatagga acttcggaat aggaactaag gaggatattc atatggacca
     3001 tggctaattc ccatgtcagc cgttaagtgt tcctgtgtca ctcaaaattg ctttgagagg
     3061 ctctaagggc ttctcagtgc gttacatccc tggcttgttg tccacaaccg ttaaacctta
     3121 aaagctttaa aagccttata tattcttttt tttcttataa aacttaaaac cttagaggct
     3181 atttaagttg ctgatttata ttaattttat tgttcaaaca tgagagctta gtacgtgaaa
     3241 catgagagct tagtacgtta gccatgagag cttagtacgt tagccatgag ggtttagttc
     3301 gttaaacatg agagcttagt acgttaaaca tgagagctta gtacgtgaaa catgagagct
     3361 tagtacgtac tatcaacagg ttgaactgcg gatcttgcgg ccgcaaaaat taaaaatgaa
     3421 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     3481 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     3541 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     3601 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     3661 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     3721 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     3781 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     3841 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     3901 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     3961 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     4021 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     4081 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     4141 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     4201 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     4261 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     4321 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     4381 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     4441 cccgaaaagt gccacctgca tcgatggccc cccgatggta gtgtggggtc tccccatgcg
     4501 agagtaggga actgccaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     4561 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccgggagc
     4621 ggatttgaac gttgcgaagc aacggcccgg agggtggcgg gcaggacgcc cgccataaac
     4681 tgccaggcat caaattaagc agaaggccat cctgacggat ggcctttttg cgtggccagt
     4741 gccaagcttg catgc
//
