LOCUS       sequence-311625-        6347 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 6347)
  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 6347)
  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-TK3
FEATURES             Location/Qualifiers
     source          1..6347
                     /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    2139..2263
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2288..2293,2294..2311,2312..2318)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2372..3031
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2438..2457)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3373..3678
                     /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     3557..3576
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3719..3843)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4088..4150
                     /label=Mini-TK promoter
                     /note="minimal herpes simplex virus (HSV) thymidine kinase 
                     promoter"
     5'UTR           4172..4211
                     /label=HSV TK 5'-UTR
                     /note="5' untranslated region from the herpes simplex virus
                     thymidine kinase gene"
     CDS             4229..5359
                     /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(5357..5376)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5401..5449
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(5753..5770)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5753..5769)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5762..5784)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5978..5997)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6097..6119
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6157..6175)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6243..6347
                     /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 cgtttgcttt
     1981 tgtttcgtgt tggtcaccac ggccgagttt cctcgggacc ccgggcagct gctttacatc
     2041 ccaaagacct atctgctcgg ccggcctcgg aacgcgagcc tgcccggaag atccccgggt
     2101 accgagctcg aattcatcgt caccatcacc tcgaatcaac aagtttgtac aaaaaagctg
     2161 aacgagaaac gtaaaatgat ataaatatca atatattaaa ttagattttg cataaaaaac
     2221 agactacata atactgtaaa acacaacata tccagtcact atggcggccg cattaggcac
     2281 cccaggcttt acactttatg cttccggctc gtataatgtg tggattttga gttaggatcc
     2341 gtcgagattt tcaggagcta aggaagctaa aatggagaaa aaaatcactg gatataccac
     2401 cgttgatata tcccaatggc atcgtaaaga acattttgag gcatttcagt cagttgctca
     2461 atgtacctat aaccagaccg ttcagctgga tattacggcc tttttaaaga ccgtaaagaa
     2521 aaataagcac aagttttatc cggcctttat tcacattctt gcccgcctga tgaatgctca
     2581 tccggaattc cgtatggcaa tgaaagacgg tgagctggtg atatgggata gtgttcaccc
     2641 ttgttacacc gttttccatg agcaaactga aacgttttca tcgctctgga gtgaatacca
     2701 cgacgatttc cggcagtttc tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa
     2761 cctggcctat ttccctaaag ggtttattga gaatatgttt ttcgtctcag ccaatccctg
     2821 ggtgagtttc accagttttg atttaaacgt ggccaatatg gacaacttct tcgcccccgt
     2881 tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc tggcgattca
     2941 ggttcatcat gccgtttgtg atggcttcca tgtcggcaga atgcttaatg aattacaaca
     3001 gtactgcgat gagtggcagg gcggggcgta aagatctgga tccggcttac taaaagccag
     3061 ataacagtat gcgtatttgc gcgctgattt ttgcggtata agaatatata ctgatatgta
     3121 tacccgaagt atgtcaaaaa gaggtatgct atgaagcagc gtattacagt gacagttgac
     3181 agcgacagct atcagttgct caaggcatat atgatgtcaa tatctccggt ctggtaagca
     3241 caaccatgca gaatgaagcc cgtcgtctgc gtgccgaacg ctggaaagcg gaaaatcagg
     3301 aagggatggc tgaggtcgcc cggtttattg aaatgaacgg ctcttttgct gacgagaaca
     3361 ggggctggtg aaatgcagtt taaggtttac acctataaaa gagagagccg ttatcgtctg
     3421 tttgtggatg tacagagtga tattattgac acgcccgggc gacggatggt gatccccctg
     3481 gccagtgcac gtctgctgtc agataaagtc tcccgtgaac tttacccggt ggtgcatatc
     3541 ggggatgaaa gctggcgcat gatgaccacc gatatggcca gtgtgccggt ctccgttatc
     3601 ggggaagaag tggctgatct cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg
     3661 atgttctggg gaatataaat gtcaggctcc cttatacaca gccagtctgc aggtcgacca
     3721 tagtgactgg atatgttgtg ttttacagta ttatgtagtc tgttttttat gcaaaatcta
     3781 atttaatata ttgatattta tatcatttta cgtttctcgt tcagctttct tgtacaaagt
     3841 ggttgattcg aggctgctaa caaatcgagc agtgtggttt tcaagaggaa gcaaaaagcc
     3901 tctccaccca ggcctggaat gtttccaccc aatgtcgagc agtgtggttt tgcaagagga
     3961 agcaaaaagc ctctccaccc aggcctggaa tgtttccacc caatgtcgag caaaccccgc
     4021 ccagcgtctt gtcattggcg aattcgaaca cgcagatgca gtcggggcgg cgcggtccga
     4081 ggtccacttc gcatattaag gtgacgcgtg tggcctcgaa caccgagcga ccctgcagcg
     4141 acccgcttaa cagcgtcaac agcgtgccgc agatcttggt ggcgtgaaac tcccgcacct
     4201 cttcggccag cgccttgtag aagcgcgtat ggcttcgtac cccggccatc aacacgcgtc
     4261 tgcgttcgac caggctgcgc gttctcgcgg ccatagcaac cgacgtacgg cgttgcgccc
     4321 tcgccggcag caagaagcca cggaagtccg cccggagcag aaaatgccca cgctactgcg
     4381 ggtttatata gacggtcccc acgggatggg gaaaaccacc accacgcaac tgctggtggc
     4441 cctgggttcg cgcgacgata tcgtctacgt acccgagccg atgacttact ggcgggtgct
     4501 gggggcttcc gagacaatcg cgaacatcta caccacacaa caccgcctcg accagggtga
     4561 gatatcggcc ggggacgcgg cggtggtaat gacaagcgcc cagataacaa tgggcatgcc
     4621 ttatgccgtg accgacgccg ttctggctcc tcatatcggg ggggaggctg ggagctcaca
     4681 tgccccgccc ccggccctca ccctcatctt cgaccgccat cccatcgccg ccctcctgtg
     4741 ctacccggcc gcgcggtacc ttatgggcag catgaccccc caggccgtgc tggcgttcgt
     4801 ggccctcatc ccgccgacct tgcccggcac caacatcgtg cttggggccc ttccggagga
     4861 cagacacatc gaccgcctgg ccaaacgcca gcgccccggc gagcggctgg acctggctat
     4921 gctggctgcg attcgccgcg tttacgggct acttgccaat acggtgcggt atctgcagtg
     4981 cggcgggtcg tggcgggagg actggggaca gctttcgggg acggccgtgc cgccccaggg
     5041 tgccgagccc cagagcaacg cgggcccacg accccatatc ggggacacgt tatttaccct
     5101 gtttcgggcc cccgagttgc tggcccccaa cggcgacctg tataacgtgt ttgcctgggc
     5161 cttggacgtc ttggccaaac gcctccgttc catgcacgtc tttatcctgg attacgacca
     5221 atcgcccgcc ggctgccggg acgccctgct gcaacttacc tccgggatgg tccagaccca
     5281 cgtcaccacc cccggctcca taccgacgat atgcgacctg gcgcgcacgt ttgcccggga
     5341 gatgggggag gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga
     5401 cggcaataaa aagacagaat aaaacgcacg ggtgttgggt cgtttgttca taaacgcggg
     5461 gttcggtccc agggctggca ctctgtcgat accccaccga gaccccattg gggccaatac
     5521 gcccgcgttt cttccttttc cccaccccac cccccaagtt cgggtgaagg cccagggctc
     5581 gcagccaacg tcggggcggc aggccctgcc atagccactg gccccgtggg ttagggacgg
     5641 ggtcccccat ggggaatggt ttatggttcg tgggggttat tattttgggc gttgcgtggg
     5701 gtcaggtcca cgacccaagc ttggctgcag gtcgagctcg cgaaagcttg gcactggccg
     5761 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag
     5821 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc
     5881 aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc cttacgcatc
     5941 tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct gatgccgcat
     6001 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc
     6061 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt
     6121 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat
     6181 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     6241 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     6301 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagt
//
