LOCUS       sequence-313502-        3144 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 3144)
  AUTHORS   Wu S, Ying G, Wu Q, Capecchi MR
  TITLE     A protocol for constructing gene targeting vectors: generating 
            knockout mice for the cadherin family and beyond.
  JOURNAL   Nat Protoc. 2008  . 3(6):1056-76.
  PUBMED    18546598
REFERENCE   2  (bases 1 to 3144)
  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: "Nat Protoc.
            2008  . 3(6):1056-76."
COMMENT     Sequence Label: pStart-K
FEATURES             Location/Qualifiers
     source          1..3144
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      290..376
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      468..495
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     503..526
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
     protein_bind    545..644
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             799..1104
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRIVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     983..1002
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(1133..1232)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     rep_origin      1365..1910
                     /direction=RIGHT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     1811..1830
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2027..2044
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     complement(2494..2513)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             join(2505..3144,1..176)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             join(2505..3144,1..176)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(2578..2597)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
ORIGIN
        1 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga
       61 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg
      121 ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag
      181 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gctagcatgg
      241 atctcgggga cgtctaacta ctaagcgaga gtagggaact gccaggcatc aaataaaacg
      301 aaaggctcag tcggaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct
      361 cctgagtagg acaaatccgc cgggagcgga tttgaacgtt gtgaagcaac ggcccggagg
      421 gtggcgggca ggacgcccgc cataaactgc caggcatcaa actaagcaga aggccatcct
      481 gacggatggc ctttttgcgt ttctacaaac tcttcctgtt agttagttac ttaagctcgg
      541 gccccaaata atgattttat tttgactgat agtgacctgt tcgttgcaac aaattgataa
      601 gcaatgcttt tttataatgc caactttgta caaaaaagca ggctttaaag gaaccaattc
      661 agtcgactgg atccggtacc gaattcgctt actaaaagcc agataacagt atgcgtattt
      721 gcgcgctgat ttttgcggta taagaatata tactgatatg tatacccgaa gtatgtcaaa
      781 aagaggtgtg cttctagaat gcagtttaag gtttacacct ataaaagaga gagccgttat
      841 cgtctgtttg tggatgtaca gagtgatatt attgacacgc ccgggcgacg gatagtgatc
      901 cccctggcca gtgcacgtct gctgtcagat aaagtctccc gtgaacttta cccggtggtg
      961 catatcgggg atgaaagctg gcgcatgatg accaccgata tggccagtgt gccggtctcc
     1021 gttatcgggg aagaagtggc tgatctcagc caccgcgaaa atgacatcaa aaacgccatt
     1081 aacctgatgt tctggggaat atagaattcg cggccgcact cgagatatct agacccagct
     1141 ttcttgtaca aagttggcat tataagaaag cattgcttat caatttgttg caacgaacag
     1201 gtcactatca gtcaaaataa aatcattatt tgccatccag ctgcagctgt caaacatgag
     1261 aattacaact tatatcgtat ggggctgact tcaggtgcta catttgaaga gataaattgc
     1321 actgaaatct agaaatattt tatctgatta ataagatgat cttcttgaga tcgttttggt
     1381 ctgcgcgtaa tctcttgctc tgaaaacgaa aaaaccgcct tgcagggcgg tttttcgaag
     1441 gttctctgag ctaccaactc tttgaaccga ggtaactggc ttggaggagc gcagtcacca
     1501 aaacttgtcc tttcagttta gccttaaccg gcgcatgact tcaagactaa ctcctctaaa
     1561 tcaattacca gtggctgctg ccagtggtgc ttttgcatgt ctttccgggt tggactcaag
     1621 acgatagtta ccggataagg cgcagcggtc ggactgaacg gggggttcgt gcatacagtc
     1681 cagcttggag cgaactgcct acccggaact gagtgtcagg cgtggaatga gacaaacgcg
     1741 gccataacag cggaatgaca ccggtaaacc gaaaggcagg aacaggagag cgcacgaggg
     1801 agccgccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc caccactgat
     1861 ttgagcgtca gatttcgtga tgcttgtcag gggggcggag cctatggaaa aacggctttg
     1921 ccgcggccct ctcacttccc tgttaagtat cttcctggca tcttccagga aatctccgcc
     1981 ccgttcgtaa gccatttccg ctcgccgcag tcgaacgacc gagcgtagcg agtcagtgag
     2041 cgaggaagcg gaatatatcc tgtatcacat attctgctga cgcaccggtg cagccttttt
     2101 tctcctgcca catgaagcac ttcactgaca ccctcatcag tgccaacata gtaagccagt
     2161 atacactccg ctagcgctga ggtctgcctc gtgaagaagg tgttgctgac tcataccagg
     2221 cctgaatcgc cccatcatcc agccagaaag tgagggagcc acggttgatg agagctttgt
     2281 tgtaggtgga ccagttggtg attttgaact tttgctttgc cacggaacgg tctgcgttgt
     2341 cgggaagatg cgtgatctga tccttcaact cagcaaaagt tcgatttatt caacaaagcc
     2401 acgttgtgtc tcaaaatctc tgatgttaca ttgcacaaga taaaaatata tcatcatgaa
     2461 caataaaact gtctgcttac ataaacagta atacaagggg tgttatgagc catattcaac
     2521 gggaaacgtc ttgctcgagg ccgcgattaa attccaacat ggatgctgat ttatatgggt
     2581 ataaatgggc tcgcgataat gtcgggcaat caggtgcgac aatctatcga ttgtatggga
     2641 agcccgatgc gccagagttg tttctgaaac atggcaaagg tagcgttgcc aatgatgtta
     2701 cagatgagat ggtcagacta aactggctga cggaatttat gcctcttccg accatcaagc
     2761 attttatccg tactcctgat gatgcatggt tactcaccac tgcgatcccc gggaaaacag
     2821 cattccaggt attagaagaa tatcctgatt caggtgaaaa tattgttgat gcgctggcag
     2881 tgttcctgcg ccggttgcat tcgattcctg tttgtaattg tccttttaac agcgatcgcg
     2941 tatttcgtct cgctcaggcg caatcacgaa tgaataacgg tttggttgat gcgagtgatt
     3001 ttgatgacga gcgtaatggc tggcctgttg aacaagtctg gaaagaaatg cataagcttt
     3061 tgccattctc accggattca gtcgtcactc atggtgattt ctcacttgat aaccttattt
     3121 ttgacgaggg gaaattaata ggtt
//
