LOCUS       sequence-311626-        6324 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 6324)
  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 6324)
  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-TK6
FEATURES             Location/Qualifiers
     source          1..6324
                     /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
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(219..238)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1026..1571
                     /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     1472..1491
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1688..1705
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     1855..1874
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1855..1873
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    2116..2240
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(2265..2270,2271..2288,2289..2295)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             2349..3008
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2415..2434)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             3350..3655
                     /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     3534..3553
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3696..3820)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4065..4127
                     /label=Mini-TK promoter
                     /note="minimal herpes simplex virus (HSV) thymidine kinase 
                     promoter"
     5'UTR           4149..4188
                     /label=HSV TK 5'-UTR
                     /note="5' untranslated region from the herpes simplex virus
                     thymidine kinase gene"
     CDS             4206..5336
                     /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(5334..5353)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5378..5426
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(5730..5747)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5730..5746)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5739..5761)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(5955..5974)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6074..6096
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(6134..6152)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6220..6324
                     /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 cgtgttgacg 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 cctgcagcaa 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 cagacccctt aataagatga
     1021 tcttcttgag atcgttttgg tctgcgcgta atctcttgct ctgaaaacga aaaaaccgcc
     1081 ttgcagggcg gtttttcgaa ggttctctga gctaccaact ctttgaaccg aggtaactgg
     1141 cttggaggag cgcagtcacc aaaacttgtc ctttcagttt agccttaacc ggcgcatgac
     1201 ttcaagacta actcctctaa atcaattacc agtggctgct gccagtggtg cttttgcatg
     1261 tctttccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cggactgaac
     1321 ggggggttcg tgcatacagt ccagcttgga gcgaactgcc tacccggaac tgagtgtcag
     1381 gcgtggaatg agacaaacgc ggccataaca gcggaatgac accggtaaac cgaaaggcag
     1441 gaacaggaga gcgcacgagg gagccgccag ggggaaacgc ctggtatctt tatagtcctg
     1501 tcgggtttcg ccaccactga tttgagcgtc agatttcgtg atgcttgtca ggggggcgga
     1561 gcctatggaa aaacggcttt gccgcggccc tctcacttcc ctgttaagta tcttcctggc
     1621 atcttccagg aaatctccgc cccgttcgta agccatttcc gctcgccgca gtcgaacgac
     1681 cgagcgtagc gagtcagtga gcgaggaagc ggaatatatc ctgtatcaca tattctgctg
     1741 acgcaccggt gcagcctttt ttctcctgcc acatgaagca cttcactgac accctcatca
     1801 gtgccaacat agtaagccag tatacactcc gctagcaacc tggcttatcg aaattaatac
     1861 gactcactat agggagaccg gcagatccgg cgcgcctgca ggcgcgccac tagttaatta
     1921 tttaaatcga tgcgacgcta gcggccgcgt ttgcttttgt ttcgtgttgg tcaccacggc
     1981 cgagtttcct cgggaccccg ggcagctgct ttacatccca aagacctatc tgctcggccg
     2041 gcctcggaac gcgagcctgc ccggaagatc cccgggtacc gagctcgaat tcatcgtcac
     2101 catcacctcg aatcaacaag tttgtacaaa aaagctgaac gagaaacgta aaatgatata
     2161 aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata ctgtaaaaca
     2221 caacatatcc agtcactatg gcggccgcat taggcacccc aggctttaca ctttatgctt
     2281 ccggctcgta taatgtgtgg attttgagtt aggatccgtc gagattttca ggagctaagg
     2341 aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc caatggcatc
     2401 gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac cagaccgttc
     2461 agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag ttttatccgg
     2521 cctttattca cattcttgcc cgcctgatga atgctcatcc ggaattccgt atggcaatga
     2581 aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt ttccatgagc
     2641 aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg cagtttctac
     2701 acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc cctaaagggt
     2761 ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc agttttgatt
     2821 taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc aaatattata
     2881 cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc gtttgtgatg
     2941 gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag tggcagggcg
     3001 gggcgtaaag atctggatcc ggcttactaa aagccagata acagtatgcg tatttgcgcg
     3061 ctgatttttg cggtataaga atatatactg atatgtatac ccgaagtatg tcaaaaagag
     3121 gtatgctatg aagcagcgta ttacagtgac agttgacagc gacagctatc agttgctcaa
     3181 ggcatatatg atgtcaatat ctccggtctg gtaagcacaa ccatgcagaa tgaagcccgt
     3241 cgtctgcgtg ccgaacgctg gaaagcggaa aatcaggaag ggatggctga ggtcgcccgg
     3301 tttattgaaa tgaacggctc ttttgctgac gagaacaggg gctggtgaaa tgcagtttaa
     3361 ggtttacacc tataaaagag agagccgtta tcgtctgttt gtggatgtac agagtgatat
     3421 tattgacacg cccgggcgac ggatggtgat ccccctggcc agtgcacgtc tgctgtcaga
     3481 taaagtctcc cgtgaacttt acccggtggt gcatatcggg gatgaaagct ggcgcatgat
     3541 gaccaccgat atggccagtg tgccggtctc cgttatcggg gaagaagtgg ctgatctcag
     3601 ccaccgcgaa aatgacatca aaaacgccat taacctgatg ttctggggaa tataaatgtc
     3661 aggctccctt atacacagcc agtctgcagg tcgaccatag tgactggata tgttgtgttt
     3721 tacagtatta tgtagtctgt tttttatgca aaatctaatt taatatattg atatttatat
     3781 cattttacgt ttctcgttca gctttcttgt acaaagtggt tgattcgagg ctgctaacaa
     3841 atcgagcagt gtggttttca agaggaagca aaaagcctct ccacccaggc ctggaatgtt
     3901 tccacccaat gtcgagcagt gtggttttgc aagaggaagc aaaaagcctc tccacccagg
     3961 cctggaatgt ttccacccaa tgtcgagcaa accccgccca gcgtcttgtc attggcgaat
     4021 tcgaacacgc agatgcagtc ggggcggcgc ggtccgaggt ccacttcgca tattaaggtg
     4081 acgcgtgtgg cctcgaacac cgagcgaccc tgcagcgacc cgcttaacag cgtcaacagc
     4141 gtgccgcaga tcttggtggc gtgaaactcc cgcacctctt cggccagcgc cttgtagaag
     4201 cgcgtatggc ttcgtacccc ggccatcaac acgcgtctgc gttcgaccag gctgcgcgtt
     4261 ctcgcggcca tagcaaccga cgtacggcgt tgcgccctcg ccggcagcaa gaagccacgg
     4321 aagtccgccc ggagcagaaa atgcccacgc tactgcgggt ttatatagac ggtccccacg
     4381 ggatggggaa aaccaccacc acgcaactgc tggtggccct gggttcgcgc gacgatatcg
     4441 tctacgtacc cgagccgatg acttactggc gggtgctggg ggcttccgag acaatcgcga
     4501 acatctacac cacacaacac cgcctcgacc agggtgagat atcggccggg gacgcggcgg
     4561 tggtaatgac aagcgcccag ataacaatgg gcatgcctta tgccgtgacc gacgccgttc
     4621 tggctcctca tatcgggggg gaggctggga gctcacatgc cccgcccccg gccctcaccc
     4681 tcatcttcga ccgccatccc atcgccgccc tcctgtgcta cccggccgcg cggtacctta
     4741 tgggcagcat gaccccccag gccgtgctgg cgttcgtggc cctcatcccg ccgaccttgc
     4801 ccggcaccaa catcgtgctt ggggcccttc cggaggacag acacatcgac cgcctggcca
     4861 aacgccagcg ccccggcgag cggctggacc tggctatgct ggctgcgatt cgccgcgttt
     4921 acgggctact tgccaatacg gtgcggtatc tgcagtgcgg cgggtcgtgg cgggaggact
     4981 ggggacagct ttcggggacg gccgtgccgc cccagggtgc cgagccccag agcaacgcgg
     5041 gcccacgacc ccatatcggg gacacgttat ttaccctgtt tcgggccccc gagttgctgg
     5101 cccccaacgg cgacctgtat aacgtgtttg cctgggcctt ggacgtcttg gccaaacgcc
     5161 tccgttccat gcacgtcttt atcctggatt acgaccaatc gcccgccggc tgccgggacg
     5221 ccctgctgca acttacctcc gggatggtcc agacccacgt caccaccccc ggctccatac
     5281 cgacgatatg cgacctggcg cgcacgtttg cccgggagat gggggaggct aactgaaaca
     5341 cggaaggaga caataccgga aggaacccgc gctatgacgg caataaaaag acagaataaa
     5401 acgcacgggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc
     5461 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc
     5521 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg
     5581 ccctgccata gccactggcc ccgtgggtta gggacggggt cccccatggg gaatggttta
     5641 tggttcgtgg gggttattat tttgggcgtt gcgtggggtc aggtccacga cccaagcttg
     5701 gctgcaggtc gagctcgcga aagcttggca ctggccgtcg ttttacaacg tcgtgactgg
     5761 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     5821 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     5881 gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     5941 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     6001 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     6061 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     6121 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     6181 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     6241 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     6301 caataatatt gaaaaaggaa gagt
//
