LOCUS       sequence-450979-        2842 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 2842)
  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 2842)
  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: pKD3
FEATURES             Location/Qualifiers
     source          1..2842
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(80..166)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        256..360
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(361..429,430..1221)
                     /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     complement(579..598)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1323..1711
                     /label=R6K gamma ori
                     /note="gamma replication origin from E. coli plasmid R6K; 
                     requires the R6K initiator protein pi for replication"
     protein_bind    1760..1793
                     /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)"
     promoter        1899..2001
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             2002..2661
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPELRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2068..2087)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     protein_bind    2678..2725
                     /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)."
     terminator      complement(2803..2830)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
ORIGIN
        1 gcctggcagt ttatggcggg cgtcctgccc gccaccctcc gggccgttgc ttcgcaacgt
       61 tcaaatccgc tcccggcgga tttgtcctac tcaggagagc gttcaccgac aaacaacaga
      121 taaaacgaaa ggcccagtct ttcgactgag cctttcattt tatttgatgc ctggcagttc
      181 cctactctcg catggggaga ccccacacta ccatcggggg ccatcgatgc aggtggcact
      241 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
      301 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
      361 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
      421 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
      481 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
      541 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
      601 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
      661 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
      721 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
      781 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
      841 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
      901 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
      961 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     1021 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
     1081 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     1141 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     1201 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     1261 ttaaaacttc atttttaatt tttgcggccg caagatccgg ccacgatgcg tccggcgtag
     1321 aggatctgaa gatcagcagt tcaacctgtt gatagtacgt actaagctct catgtttcac
     1381 gtactaagct ctcatgttta acgtactaag ctctcatgtt taacgaacta aaccctcatg
     1441 gctaacgtac taagctctca tggctaacgt actaagctct catgtttcac gtactaagct
     1501 ctcatgtttg aacaataaaa ttaatataaa tcagcaactt aaatagcctc taaggtttta
     1561 agttttataa gaaaaaaaag aatatataag gcttttaaag cttttaaggt ttaacggttg
     1621 tggacaacaa gccagggatg taacgcactg agaagccctt agagcctctc aaagcaattt
     1681 tgagtgacac aggaacactt aacggctgac atgggaatta gccatggtcc atatgaatat
     1741 cctccttagt tcctattccg aagttcctat tctctagaaa gtataggaac ttcggcgcgc
     1801 ctacctgtga cggaagatca cttcgcagaa taaataaatc ctggtgtccc tgttgatacc
     1861 gggaagccct gggccaactt ttggcgaaaa tgagacgttg atcggcacgt aagaggttcc
     1921 aactttcacc ataatgaaat aagatcacta ccgggcgtat tttttgagtt gtcgagattt
     1981 tcaggagcta aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata
     2041 tcccaatggc atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat
     2101 aaccagaccg ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac
     2161 aagttttatc cggcctttat tcacattctt gcccgcctga tgaatgctca tccggaatta
     2221 cgtatggcaa tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc
     2281 gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc
     2341 cggcagtttc tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat
     2401 ttccctaaag ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc
     2461 accagttttg atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg
     2521 ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat
     2581 gccgtttgtg atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat
     2641 gagtggcagg gcggggcgta aggcgcgcca tttaaatgaa gttcctattc cgaagttcct
     2701 attctctaga aagtatagga acttcgaagc agctccagcc tacacaatcg ctcaagacgt
     2761 gtaatgctgc aatctgcatg caagcttggc actggccacg caaaaaggcc atccgtcagg
     2821 atggccttct gcttaatttg at
//
