LOCUS       sequence-311628-        8473 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 8473)
  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 8473)
  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: pWS-TK2
FEATURES             Location/Qualifiers
     source          1..8473
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             join(1..69,70..861)
                     /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(219..238)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1032..1620
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1521..1540
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1774..1791
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1878..1897
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1878..1896
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    4265..4389
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(4414..4419,4420..4437,4438..4444)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             4498..5157
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(4564..4583)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             5499..5804
                     /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="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     5683..5702
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(5845..5969)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        6214..6276
                     /label=Mini-TK promoter
                     /note="minimal herpes simplex virus (HSV) thymidine kinase 
                     promoter"
     5'UTR           6298..6337
                     /label=HSV TK 5'-UTR
                     /note="5' untranslated region from the herpes simplex virus
                     thymidine kinase gene"
     CDS             6355..7485
                     /codon_start=1
                     /product="herpes simplex virus thymidine kinase"
                     /label=HSV TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MASYPGHQHASAFDQAARSRGHSNRRTALRPRRQQEATEVRPEQK
                     MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRVLGASETIANIYTTQ
                     HRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGSSHAPPPALTLIFDR
                     HPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGALPEDRHIDRLAKRQR
                     PGERLDLAMLAAIRRVYGLLANTVRYLQCGGSWREDWGQLSGTAVPPQGAEPQSNAGPR
                     PHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRSMHVFILDYDQSPAGCRDAL
                     LQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
     primer_bind     complement(7483..7502)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7527..7575
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(7879..7896)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7879..7895)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7888..7910)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(8104..8123)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     8223..8245
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(8283..8301)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        8369..8473
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
       61 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
      121 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
      181 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
      241 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
      301 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
      361 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
      421 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
      481 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
      541 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
      601 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
      661 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtgggtct
      721 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
      781 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
      841 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
      901 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
      961 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     1021 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     1081 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     1141 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     1201 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     1261 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     1321 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     1381 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     1441 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     1501 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     1561 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     1621 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     1681 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     1741 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     1801 gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc cgattcatta atgcaggtta
     1861 acctggctta tcgaaattaa tacgactcac tatagggaga ccggcagatc cggcgcgcct
     1921 gcaggcgcgc cactagttaa ttatttaaat cgatgcgacg ctagcggccg cgtttaaacg
     1981 gccctcgact ctagtcgaga tcctctagtc gagcagtgtg gttttcaaga ggaagcaaaa
     2041 agcctctcca cccaggcctg gaatgtttcc acccaatgtc gagcagtgtg gttttgcaag
     2101 aggaagcaaa aagcctctcc acccaggcct ggaatgtttc cacccaatgt cgagcaaacc
     2161 ccgcccagcg tcttgtcatt ggcgaattcg aacacgcaga tgcagtctgg gcggcgcggc
     2221 ccgaggtcca cttcgcatat taaggtgacg cgcgtggcct cgaacagcga gcgaccctgc
     2281 agcgacccgc tcatcagcgt cagcagcgtt ccacaaatcc tggtggcgtt gaactcccgc
     2341 acctctcggg cgaacgcctt gtagaagcgg gtatggcttc tcacgccggc caacagcacg
     2401 cgcctgcgtt cggtcaggct gctcgtgcga gcgggcctac cgacggccgc gcggcgtccc
     2461 gtcctagcca tcgccagggg gcctccgaag cccgcgggga tccggagctg cccacgctgc
     2521 tgcgggttta tatagacgga ccccacgggg tggggaagac caccacctcc gcgcagctga
     2581 tggaggccct ggggccgcgc gacaatatcg tctacgtccc cgagccgatg acttactggc
     2641 aggtgctggg ggcctccgag accctgacga acatctacaa cacgcagcac cgtctggacc
     2701 gcggcgagat atcggccggg gaggcggcgg tggtaatgac cagcgcccag ataacaatga
     2761 gcacgcctta tgcggcgacg gacgccgttt tggctcctca tatcgggggg gaggctgtgg
     2821 gcccgcaagc cccgcccccg gccctcaccc ttgttttcga ccggcaccct atcgcctccc
     2881 tgctgtgcta cccggccgcg cggtacctca tgggaagcat gaccccccag gccgtgttgg
     2941 cgttcgtggc cctcatgccc ccgaccgcgc ccggcacgaa cctggtcctg ggtgtccttc
     3001 cggaggccga acacgccgac cgcctggcca gacgccaacg cccgggcgag cggcttgacc
     3061 tggccatgct gtccgccatt cgccgtgtct acgatctact cgccaacacg gtgcggtacc
     3121 tgcagcgcgg cgggaggtgg cgggaggact ggggccggct gacgggggtc gccgcggcga
     3181 ccccgcgccc cgaccccgag gacggcgcgg ggtctctgcc ccgcatcgag gacacgctgt
     3241 ttgccctgtt ccgcgttccc gagctgctgg cccccaacgg ggacttgtac cacatttttg
     3301 cctgggtctt ggacgtcttg gccgaccgcc tccttccgat gcatctattt gtcctggatt
     3361 acgatcagtc gcccgtcggg tgtcgagacg ccctgttgcg cctcaccgcc gggatgatcc
     3421 caacccgcgt cacaaccgcc gggtccatcg ccgagatacg cgacctggcg cgcacgtttg
     3481 cccgcgaggt ggggggagtt tagttcaaac acggaagccc gaacggaagg cctcccggcg
     3541 atgacggcaa taaaagaaca gaataaaagg cattgttgtc gtgtggtgtg tccataagcg
     3601 cgggggttcg gggccagggc tggcaccgta tcagcacccc accgaaaaac ggagcgggcc
     3661 gatccgtcct tgttttcggt ctggtactcc ctttgtgctt ttaccctcac cccaccccat
     3721 cctttggccc gcgcttacgg caacaaaggg cctccgatag cctccgaggt gcggagcctc
     3781 tttgggccgt gggtacggac acccccccat ctgcggactg gcagccggga cggacgacca
     3841 tgggccccgg tctgtgggtg gtgatggggg tcctggtggg cgttgccggg ggccatgaca
     3901 cgtactggac ggagcaaatc gacccgtggt ttttgcacgg tctggggttg gcccgcacgt
     3961 actggcgcga cacaaacacc gggcgtctgt ggttgcccaa cacccccgac gccagcgacc
     4021 cccagcgcgg acgcttggcg cccccgggcg aactcaacct gactacggca tccgtgccca
     4081 tgcttcggtg gtacgccgag cgcttttgtt tcgtgttggt caccacggcc gagtttcctc
     4141 gggaccccgg gcagctgctt tacatcccaa agacctatct gctcggccgg cctcggaacg
     4201 cgagcctgcc cggaagatcc ccgggtaccg agctcgaatt catcgtcacc atcacctcga
     4261 atcaacaagt ttgtacaaaa aagctgaacg agaaacgtaa aatgatataa atatcaatat
     4321 attaaattag attttgcata aaaaacagac tacataatac tgtaaaacac aacatatcca
     4381 gtcactatgg cggccgcatt aggcacccca ggctttacac tttatgcttc cggctcgtat
     4441 aatgtgtgga ttttgagtta ggatccgtcg agattttcag gagctaagga agctaaaatg
     4501 gagaaaaaaa tcactggata taccaccgtt gatatatccc aatggcatcg taaagaacat
     4561 tttgaggcat ttcagtcagt tgctcaatgt acctataacc agaccgttca gctggatatt
     4621 acggcctttt taaagaccgt aaagaaaaat aagcacaagt tttatccggc ctttattcac
     4681 attcttgccc gcctgatgaa tgctcatccg gaattccgta tggcaatgaa agacggtgag
     4741 ctggtgatat gggatagtgt tcacccttgt tacaccgttt tccatgagca aactgaaacg
     4801 ttttcatcgc tctggagtga ataccacgac gatttccggc agtttctaca catatattcg
     4861 caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc ctaaagggtt tattgagaat
     4921 atgtttttcg tctcagccaa tccctgggtg agtttcacca gttttgattt aaacgtggcc
     4981 aatatggaca acttcttcgc ccccgttttc accatgggca aatattatac gcaaggcgac
     5041 aaggtgctga tgccgctggc gattcaggtt catcatgccg tttgtgatgg cttccatgtc
     5101 ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt ggcagggcgg ggcgtaaaga
     5161 tctggatccg gcttactaaa agccagataa cagtatgcgt atttgcgcgc tgatttttgc
     5221 ggtataagaa tatatactga tatgtatacc cgaagtatgt caaaaagagg tatgctatga
     5281 agcagcgtat tacagtgaca gttgacagcg acagctatca gttgctcaag gcatatatga
     5341 tgtcaatatc tccggtctgg taagcacaac catgcagaat gaagcccgtc gtctgcgtgc
     5401 cgaacgctgg aaagcggaaa atcaggaagg gatggctgag gtcgcccggt ttattgaaat
     5461 gaacggctct tttgctgacg agaacagggg ctggtgaaat gcagtttaag gtttacacct
     5521 ataaaagaga gagccgttat cgtctgtttg tggatgtaca gagtgatatt attgacacgc
     5581 ccgggcgacg gatggtgatc cccctggcca gtgcacgtct gctgtcagat aaagtctccc
     5641 gtgaacttta cccggtggtg catatcgggg atgaaagctg gcgcatgatg accaccgata
     5701 tggccagtgt gccggtctcc gttatcgggg aagaagtggc tgatctcagc caccgcgaaa
     5761 atgacatcaa aaacgccatt aacctgatgt tctggggaat ataaatgtca ggctccctta
     5821 tacacagcca gtctgcaggt cgaccatagt gactggatat gttgtgtttt acagtattat
     5881 gtagtctgtt ttttatgcaa aatctaattt aatatattga tatttatatc attttacgtt
     5941 tctcgttcag ctttcttgta caaagtggtt gattcgaggc tgctaacaaa tcgagcagtg
     6001 tggttttcaa gaggaagcaa aaagcctctc cacccaggcc tggaatgttt ccacccaatg
     6061 tcgagcagtg tggttttgca agaggaagca aaaagcctct ccacccaggc ctggaatgtt
     6121 tccacccaat gtcgagcaaa ccccgcccag cgtcttgtca ttggcgaatt cgaacacgca
     6181 gatgcagtcg gggcggcgcg gtccgaggtc cacttcgcat attaaggtga cgcgtgtggc
     6241 ctcgaacacc gagcgaccct gcagcgaccc gcttaacagc gtcaacagcg tgccgcagat
     6301 cttggtggcg tgaaactccc gcacctcttc ggccagcgcc ttgtagaagc gcgtatggct
     6361 tcgtaccccg gccatcaaca cgcgtctgcg ttcgaccagg ctgcgcgttc tcgcggccat
     6421 agcaaccgac gtacggcgtt gcgccctcgc cggcagcaag aagccacgga agtccgcccg
     6481 gagcagaaaa tgcccacgct actgcgggtt tatatagacg gtccccacgg gatggggaaa
     6541 accaccacca cgcaactgct ggtggccctg ggttcgcgcg acgatatcgt ctacgtaccc
     6601 gagccgatga cttactggcg ggtgctgggg gcttccgaga caatcgcgaa catctacacc
     6661 acacaacacc gcctcgacca gggtgagata tcggccgggg acgcggcggt ggtaatgaca
     6721 agcgcccaga taacaatggg catgccttat gccgtgaccg acgccgttct ggctcctcat
     6781 atcggggggg aggctgggag ctcacatgcc ccgcccccgg ccctcaccct catcttcgac
     6841 cgccatccca tcgccgccct cctgtgctac ccggccgcgc ggtaccttat gggcagcatg
     6901 accccccagg ccgtgctggc gttcgtggcc ctcatcccgc cgaccttgcc cggcaccaac
     6961 atcgtgcttg gggcccttcc ggaggacaga cacatcgacc gcctggccaa acgccagcgc
     7021 cccggcgagc ggctggacct ggctatgctg gctgcgattc gccgcgttta cgggctactt
     7081 gccaatacgg tgcggtatct gcagtgcggc gggtcgtggc gggaggactg gggacagctt
     7141 tcggggacgg ccgtgccgcc ccagggtgcc gagccccaga gcaacgcggg cccacgaccc
     7201 catatcgggg acacgttatt taccctgttt cgggcccccg agttgctggc ccccaacggc
     7261 gacctgtata acgtgtttgc ctgggccttg gacgtcttgg ccaaacgcct ccgttccatg
     7321 cacgtcttta tcctggatta cgaccaatcg cccgccggct gccgggacgc cctgctgcaa
     7381 cttacctccg ggatggtcca gacccacgtc accacccccg gctccatacc gacgatatgc
     7441 gacctggcgc gcacgtttgc ccgggagatg ggggaggcta actgaaacac ggaaggagac
     7501 aataccggaa ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacgggtg
     7561 ttgggtcgtt tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc
     7621 caccgagacc ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc
     7681 caagttcggg tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag
     7741 ccactggccc cgtgggttag ggacggggtc ccccatgggg aatggtttat ggttcgtggg
     7801 ggttattatt ttgggcgttg cgtggggtca ggtccacgac ccaagcttgg ctgcaggtcg
     7861 agctcgcgaa agcttggcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     7921 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     7981 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct
     8041 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct
     8101 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     8161 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     8221 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     8281 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     8341 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     8401 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     8461 aaaaaggaag agt
//
