LOCUS       sequence-63916-e        3267 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3267)
  AUTHORS   Datsenko KA, Wanner BL
  TITLE     One-step inactivation of chromosomal genes in Escherichia coli K-12 
            using PCR products.
  JOURNAL   Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5.
  PUBMED    10829079
REFERENCE   2  (bases 1 to 3267)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2000-06-6"; volume: "97(12)"; pages: 
            "6640-5"
COMMENT     Sequence Label: pKD4
FEATURES             Location/Qualifiers
     source          1..3267
                     /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"
     protein_bind    complement(1444..1477)
                     /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      1526..1882
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     CDS             complement(join(1979..2770,2771..2839))
                     /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     2602..2621
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2840..2944)
                     /gene="bla"
                     /label=AmpR promoter
     terminator      3035..3121
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3213..3240
                     /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 tcaaaagcgc
     1441 tctgaagttc ctatactttc tagagaatag gaacttcgga ataggaacta aggaggatat
     1501 tcatatggac catggctaat tcccatgtca gccgttaagt gttcctgtgt cactgaaaat
     1561 tgctttgaga ggctctaagg gcttctcagt gcgttacatc cctggcttgt tgtccacaac
     1621 cgttaaacct taaaagcttt aaaagcctta tatattcttt tttttcttat aaaacttaaa
     1681 accttagagg ctatttaagt tgctgattta tattaatttt attgttcaaa catgagagct
     1741 tagtacgtga aacatgagag cttagtacgt tagccatgag agcttagtac gttagccatg
     1801 agggtttagt tcgttaaaca tgagagctta gtacgttaaa catgagagct tagtacgtga
     1861 aacatgagag cttagtacgt actatcaaca ggttgaactg cggatcttgc ggccgcaaaa
     1921 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     1981 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     2041 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     2101 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     2161 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     2221 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     2281 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     2341 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     2401 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     2461 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     2521 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     2581 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     2641 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     2701 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     2761 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     2821 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     2881 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     2941 cgcgcacatt tccccgaaaa gtgccacctg catcgatggc cccccgatgg tagtgtgggg
     3001 tctccccatg cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa
     3061 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     3121 tccgccggga gcggatttga acgttgcgaa gcaacggccc ggagggtggc gggcaggacg
     3181 cccgccataa actgccaggc atcaaattaa gcagaaggcc atcctgacgg atggcctttt
     3241 tgcgtggcca gtgccaagct tgcatgc
//
