LOCUS       sequence-311609-        3048 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 3048)
  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 3048)
  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-C2
FEATURES             Location/Qualifiers
     source          1..3048
                     /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"
     rep_origin      822..1367
                     /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     1268..1287
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1484..1501
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        1893..1995
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1996..2655
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2062..2081)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2795..2881
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     terminator      2973..3000
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     primer_bind     3008..3031
                     /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 cagctgtcaa acatgagaat
      721 tacaacttat atcgtatggg gctgacttca ggtgctacat ttgaagagat aaattgcact
      781 gaaatctaga aatattttat ctgattaata agatgatctt cttgagatcg ttttggtctg
      841 cgcgtaatct cttgctctga aaacgaaaaa accgccttgc agggcggttt ttcgaaggtt
      901 ctctgagcta ccaactcttt gaaccgaggt aactggcttg gaggagcgca gtcaccaaaa
      961 cttgtccttt cagtttagcc ttaaccggcg catgacttca agactaactc ctctaaatca
     1021 attaccagtg gctgctgcca gtggtgcttt tgcatgtctt tccgggttgg actcaagacg
     1081 atagttaccg gataaggcgc agcggtcgga ctgaacgggg ggttcgtgca tacagtccag
     1141 cttggagcga actgcctacc cggaactgag tgtcaggcgt ggaatgagac aaacgcggcc
     1201 ataacagcgg aatgacaccg gtaaaccgaa aggcaggaac aggagagcgc acgagggagc
     1261 cgccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac cactgatttg
     1321 agcgtcagat ttcgtgatgc ttgtcagggg ggcggagcct atggaaaaac ggctttgccg
     1381 cggccctctc acttccctgt taagtatctt cctggcatct tccaggaaat ctccgccccg
     1441 ttcgtaagcc atttccgctc gccgcagtcg aacgaccgag cgtagcgagt cagtgagcga
     1501 ggaagcggaa tatatcctgt atcacatatt ctgctgacgc accggtgcag ccttttttct
     1561 cctgccacat gaagcacttc actgacaccc tcatcagtgc caacatagta agccagtata
     1621 cactccgcta gcgctgatgt ccggcggtgc ttttgccgtt acgcaccacc ccgtcagtag
     1681 ctgaacagga gggacagctg atagaaacag aagccactgg agcacctcaa aaacaccatc
     1741 atacactaaa tcagtaagtt ggcagcatca cccgacgcac tttgcgccga ataaatacct
     1801 gtgacggaag atcacttcgc agaataaata aatcctggtg tccctgttga taccgggaag
     1861 ccctgggcca acttttggcg aaaatgagac gttgatcggc acgtaagagg ttccaacttt
     1921 caccataatg aaataagatc actaccgggc gtattttttg agttatcgag attttcagga
     1981 gctaaggaag ctaaaatgga gaaaaaaatc actggatata ccaccgttga tatatcccaa
     2041 tggcatcgta aagaacattt tgaggcattt cagtcagttg ctcaatgtac ctataaccag
     2101 accgttcagc tggatattac ggccttttta aagaccgtaa agaaaaataa gcacaagttt
     2161 tatccggcct ttattcacat tcttgcccgc ctgatgaatg ctcatccgga attccgtatg
     2221 gcaatgaaag acggtgagct ggtgatatgg gatagtgttc acccttgtta caccgttttc
     2281 catgagcaaa ctgaaacgtt ttcatcgctc tggagtgaat accacgacga tttccggcag
     2341 tttctacaca tatattcgca agatgtggcg tgttacggtg aaaacctggc ctatttccct
     2401 aaagggttta ttgagaatat gtttttcgtc tcagccaatc cctgggtgag tttcaccagt
     2461 tttgatttaa acgtggccaa tatggacaac ttcttcgccc ccgttttcac catgggcaaa
     2521 tattatacgc aaggcgacaa ggtgctgatg ccgctggcga ttcaggttca tcatgccgtt
     2581 tgtgatggct tccatgtcgg cagaatgctt aatgaattac aacagtactg cgatgagtgg
     2641 cagggcgggg cgtaattttt ttaaggcagt tattggtgcc cttaaacatg ttctttcctg
     2701 cgttatcccc tgattctgtg gataaccgta ttaccgctag catggatctc ggggacgtct
     2761 aactactaag cgagagtagg gaactgccag gcatcaaata aaacgaaagg ctcagtcgga
     2821 agactgggcc tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa
     2881 tccgccggga gcggatttga acgttgtgaa gcaacggccc ggagggtggc gggcaggacg
     2941 cccgccataa actgccaggc atcaaactaa gcagaaggcc atcctgacgg atggcctttt
     3001 tgcgtttcta caaactcttc ctgttagtta gttacttaag ctcgggcc
//
