LOCUS       sequence-311631-        3265 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 3265)
  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 3265)
  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-T2
FEATURES             Location/Qualifiers
     source          1..3265
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    2..101
                     /gene="mutant version of attL"
                     /label=attL1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             256..561
                     /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     440..459
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(590..689)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(712..1902)
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     1531..1550
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        complement(join(1950..1955,1956..1972,1973..1978))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     rep_origin      2090..2635
                     /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     2536..2555
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2752..2769
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      3012..3098
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      3190..3217
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     3225..3248
                     /label=pENTR-F
                     /note="pENTR vectors, forward primer"
ORIGIN
        1 ccaaataatg attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgataagca
       61 atgctttttt ataatgccaa ctttgtacaa aaaagcaggc tttaaaggaa ccaattcagt
      121 cgactggatc cggtaccgaa ttcgcttact aaaagccaga taacagtatg cgtatttgcg
      181 cgctgatttt tgcggtataa gaatatatac tgatatgtat acccgaagta tgtcaaaaag
      241 aggtgtgctt ctagaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt
      301 ctgtttgtgg atgtacagag tgatattatt gacacgcccg ggcgacggat agtgatcccc
      361 ctggccagtg cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat
      421 atcggggatg aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt
      481 atcggggaag aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac
      541 ctgatgttct ggggaatata gaattcgcgg ccgcactcga gatatctaga cccagctttc
      601 ttgtacaaag ttggcattat aagaaagcat tgcttatcaa tttgttgcaa cgaacaggtc
      661 actatcagtc aaaataaaat cattatttgc catccagctg caggcttcca ttcaggtcga
      721 ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt atagggcggc
      781 gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa tcgccgtgac
      841 gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc cttgaagctg
      901 tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg gcatcccgat
      961 gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg tcgcgaacgc
     1021 cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct gcttctcgcc
     1081 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     1141 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     1201 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     1261 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     1321 caagggcatc ggtcgacgct ctcccttatg cgactcctgc attaggaagc agcccagtag
     1381 taggttgagg ccgttgagca ccgccgccgc aaggaatggt gcatgcaagg agatggcgcc
     1441 caacagtccc ccggccacgg ggcctgccac catacccacg ccgaaacaag cgctcatgag
     1501 cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg gcgatatagg cgccagcaac
     1561 cgcacctgtg gcgccggtga tgccggccac gatgcgtccg gcgtagagga tccacaggac
     1621 gggtgtggtc gccatgatcg cgtagtcgat agtggctcca agtagcgaag cgagcaggac
     1681 tgggcggcgg ccaaagcggt cggacagtgc tccgagaacg ggtgcgcata gaaattgcat
     1741 caacgcatat agcgctagca gcacgccata gtgactggcg atgctgtcgg aatggacgat
     1801 atcccgcaag aggcccggca gtaccggcat aaccaagcct atgcctacag catccagggt
     1861 gacggtgccg aggatgacga tgagcgcatt gttagatttc atacacggtg cctgactgcg
     1921 ttagcaattt aactgtgata aactaccgca ttaaagctta tcgatgataa gctgtcaaac
     1981 atgagaatta caacttatat cgtatggggc tgacttcagg tgctacattt gaagagataa
     2041 attgcactga aatctagaaa tattttatct gattaataag atgatcttct tgagatcgtt
     2101 ttggtctgcg cgtaatctct tgctctgaaa acgaaaaaac cgccttgcag ggcggttttt
     2161 cgaaggttct ctgagctacc aactctttga accgaggtaa ctggcttgga ggagcgcagt
     2221 caccaaaact tgtcctttca gtttagcctt aaccggcgca tgacttcaag actaactcct
     2281 ctaaatcaat taccagtggc tgctgccagt ggtgcttttg catgtctttc cgggttggac
     2341 tcaagacgat agttaccgga taaggcgcag cggtcggact gaacgggggg ttcgtgcata
     2401 cagtccagct tggagcgaac tgcctacccg gaactgagtg tcaggcgtgg aatgagacaa
     2461 acgcggccat aacagcggaa tgacaccggt aaaccgaaag gcaggaacag gagagcgcac
     2521 gagggagccg ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacca
     2581 ctgatttgag cgtcagattt cgtgatgctt gtcagggggg cggagcctat ggaaaaacgg
     2641 ctttgccgcg gccctctcac ttccctgtta agtatcttcc tggcatcttc caggaaatct
     2701 ccgccccgtt cgtaagccat ttccgctcgc cgcagtcgaa cgaccgagcg tagcgagtca
     2761 gtgagcgagg aagcggaata tatcctgtat cacatattct gctgacgcac cggtgcagcc
     2821 ttttttctcc tgccacatga agcacttcac tgacaccctc atcagtgcca acatagtaag
     2881 ccagtataca ctccgctagc gcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2941 aaccgtatta ccgctagcat ggatctcggg gacgtctaac tactaagcga gagtagggaa
     3001 ctgccaggca tcaaataaaa cgaaaggctc agtcggaaga ctgggccttt cgttttatct
     3061 gttgtttgtc ggtgaacgct ctcctgagta ggacaaatcc gccgggagcg gatttgaacg
     3121 ttgtgaagca acggcccgga gggtggcggg caggacgccc gccataaact gccaggcatc
     3181 aaactaagca gaaggccatc ctgacggatg gcctttttgc gtttctacaa actcttcctg
     3241 ttagttagtt acttaagctc gggcc
//
