LOCUS       sequence-491942-        7888 bp    DNA     circular SYN 05-NOV-2025
DEFINITION  Expression vector for mouse Fzd4, N-terminal CLIPf tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7888)
  AUTHORS   Vallon M, Yuki K, Nguyen TD, Chang J, Yuan J, Siepe D, Miao Y, 
            Essler M, Noda M, Garcia KC, Kuo CJ
  TITLE     A RECK-WNT7 Receptor-Ligand Interaction Enables Isoform-Specific 
            Regulation of Wnt Bioavailability.
  JOURNAL   Cell Rep. 2018 Oct 9;25(2):339-349.e9. doi: 
            10.1016/j.celrep.2018.09.045.
  PUBMED    30304675
REFERENCE   2  (bases 1 to 7888)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 5, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2018.09"; journalName: "Cell Rep"; date: 
            "2018-10-9- 9"; volume: "25"; issue: "2"; pages: "339-349.e9"
COMMENT     Sequence Label: pRK5-CLIPf-Fzd4
FEATURES             Location/Qualifiers
     source          1..7888
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(2527..3072)
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylcytosine 
                     derivatives"
                     /label=CLIP-tag(TM)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="MDKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVE
                     VPAPAAVLGGPEPLIQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLK
                     VVKFGEVISESHLAALVGNPAATAAVNTALDGNPVPILIPCHRVVQGDSDVGPYLGGLA
                     VKEWLLAHEGHRLGKPGLG"
     primer_bind     complement(3241..3260)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     promoter        complement(3273..3291)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3274..3291)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3501..3525)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(3522..3725)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(3551..3571)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        3726..4105
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(4134..4150)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4134..4150)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4147..4169)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4158..4174
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4182..4188,4189..4206,4207..4212))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4227..4248
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4365..4382)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4536..5124)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4616..4635)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5295..6086,6087..6155))
                     /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     5918..5937
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6156..6260)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6328..6346
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6384..6406)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6506..6525
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(6542..6997)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6679..6700)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6891..6910
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7195..7217
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7209..7226
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7210..7226
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(7246..7603)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      7260..7395
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        7412..7603
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     7560..7580
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    7672..7806
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7696..7715)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7750..7769
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     regulatory      7810..7819
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 ttttaattgt aattgacaca cacaaattac acagttaatc tgtgtgaatg caaagttgcc
       61 aagaaattta acatttcagt tcttccagac tggcgtctgc ctagatgcaa tcacacaaac
      121 taaaatattt taagtcccaa ttgcttaaca ttgcttaatg caataggtct ctagggtcgg
      181 taaggtaagt caacttagca aatctcattt ttctctgaat taagtgattg tcagaaagtg
      241 aatcaccttc ccttttcctc tcattaggca tgctgcagtg caaaaaaaaa tcctataaaa
      301 ataacctttt tttttttgag tcattagttt aaagtgaaga aaacaggggc tgcctagaat
      361 tagggatgtt gaaatttcag gagagagaaa tccactggat accttacgtg tgttagtaca
      421 gtacttttgt ttgctcaaac aattctgttt tacgacaaag gcactgaagg attttgacac
      481 cacctcttgg gagtgcgcta cagagttcta aacagcagac agtgtaggcc agaagatgtt
      541 tatgttagac cagccaaact ataaaactct gcaacacgtc tcccaaccca gcatttccag
      601 tgtttataga tcactttgct atctgcaaga cctagttact atttaaaatt taaaagggtt
      661 ttaaaaatta atctttctcc ttgcatgtta tcaactcaca gttgaaatcc cacccagtag
      721 gagcaaacac tatcaccctg ggcttcagga atctaggcgg gacctccacg gactccaagc
      781 tgtgacctgc taaagtgatc aacattttga tttttcacag ctttgaaaac ctctaactgg
      841 cttttccatt ttggaccatt ccaaagtctg cagtgaagcc attggtcttc tgatgctgag
      901 ttgggtgagt ggtggtgggc tgacgtgggt gtgtgtgggc tgaagtgttt ctaagtttgt
      961 tcaaactgag attccctgca tcacttcctt cacaatgagg aacgaggaag ccggctagtc
     1021 ttataccaca gtctcgttgc cttttcctgg cttcacccaa ccattccccc tcttctctct
     1081 ctttaccttc ccagaattca ccaatcggtt agaacacttt tgccacgtgt gaagagtttt
     1141 ggcagaccaa atccacatgc ctgaagtgat gcccacgagc aaagacataa aaattttcaa
     1201 catttcaact gccatgtttg agtcatctgc agaatatcga aagagtgccc agtttgagat
     1261 ttcatagaaa taacaggcaa tcacacaggt tgcaggaacc gtgtacagta ctgagaagac
     1321 cccgatcttg accattagcc tttccaactt gtctgtcttt gtcccgtctt tttgaagatt
     1381 ggaccgaatt ttgaataagg ccaccaaacc cgccgcaatg aacagcgttc caatcaccaa
     1441 atacgtaaag agaggagcca ccacaaagcc agtgagggca tctaggtttt ggttcccaac
     1501 atagcacaag ccagtcagtt catcggcatc cactagtctc ataatcaaga tgacaatggt
     1561 tttcactgcg ggaatagccc aggctgcgat gtggaaataa gaactgtgca tttcaatggc
     1621 ttcatgaccc cacttgagtc cggctgccaa aaaccaagtg agtgtcagaa taacccacca
     1681 aatggagctg gccattccaa aaaagtacat cagcaagaaa attattgcac atcctgtgtt
     1741 cttaagtcct tcttggatga gaacgggctc tgccgcctct tcaaaatcac aggatatcct
     1801 ttcccggcct acagtcagcc gaacaatata agcaatgcta taaatattat agcacatact
     1861 gagaaatatg atggggcgct cagggtaaga aaacctggat gaatcaatca ggaaggtcag
     1921 cacggtgaag gtggtggaga tgaagcagag gctggcccac acagccatcc aaatatccgt
     1981 gaactcctta gctgagcggc tgtacaagcc agcatcgtag ccacacttga gaacacagtt
     2041 caggctcctc ttcacccaga tgtactgatc agaattgctt cccacggagt ggcactcttc
     2101 cccgggctgg atgggagtct tgtggggcaa gggaacctct tcatcacctg gtccttccat
     2161 gcacatgtgg ttgtggtcgt tctggggcgg gaacttgctg cagttcaggg tgtcgggcca
     2221 ggcaaaccca aattctctca ggactggttc acagcgtctc ttgactgaaa ggcacatgcc
     2281 accgcacggg ccgatgggga tgttgatctt ctctgtgcac attggcacat aaaccgaaca
     2341 aaggaagaac tgcagctggc tggagcagcc gtactggatg agcggcgtga aagttgtcag
     2401 ctgcagctcg gcgtctgtct gcagctcgtg tcccactaag ttgggcatct tggtcacgtt
     2461 gtagccgagg ttctggcaca tggcgatgcg gatggggtcg cagcgccgct cctcctcgtc
     2521 cccgaaaccc agcccaggct tgcccagtct gtggccctcg tgggccagca gccactcttt
     2581 cacggcgagc ccgcccaggt agggccccac gtcgctgtcg ccctgcacca cccggtggca
     2641 ggggatcaga atgggcacgg gatttccgtc cagggcggtg ttcacggcgg cggtggcggc
     2701 gggattgccc accagggcgg ccaggtggct ctcgctgatg acctctccga acttcaccac
     2761 tttcagcagt ttccacagca cctggcgggt aaagctctcc tgctggaaca ctgggtggtg
     2821 cagggctggc acagggaact cctcgatggc ctcaggctgg tgaaagtagg cgttgagcca
     2881 ggcggtggcc tggatcagtg gctctggtcc gcccagcacg gcggctgggg caggcacttc
     2941 cacggcgtcg gcggcagatg ttcctttgcc caggaagatg atacggtgca ggccctgttc
     3001 gcacccagac agttccagct tgcccagagg gctatccagg gtggtgcgct tcatttcgca
     3061 gtctttgtcc atccccagtg tcggccgcag gagcaggagg aactgcagca gcagccccag
     3121 cctgagcccg acgcctccag gcgccccccg gctgctcggc cctgtgcccg gccaggccat
     3181 ggtggatccc cggggaattc aatcgataga accgaggtgc agttggacct gggagtggac
     3241 acctgtggag agaaaggcaa agtggatgtt attctatagt gtcacctaaa tcgtatgtgt
     3301 atgatacata aggttatgta ttaattgtag ccgcgttcta acgaagccaa gggggtgggc
     3361 ctatagactc tataggcggt acttacgtca ctcttggcac ggggaatccg cgttccaatg
     3421 caccgttccc ggccgcggag gctggatcgg tcccggtgtc ttctatggag gtcaaaacag
     3481 cgtggatggc gtctccaggc gatctgacgg ttcactaaac gagctctgct tatatagacc
     3541 tcccaccgta cacgcctacc gcccatttgc gtcaatgggg cggagttgtt acgacatttt
     3601 ggaaagtccc gttgattttg gtgccaaaac aaactcccat tgacgtcaat ggggtggaga
     3661 cttggaaatc cccgtgagtc aaaccgctat ccacgcccat tgatgtactg ccaaaaccgc
     3721 atcaccatgg taatagcgat gactaatacg tagatgtact gccaagtagg aaagtcccat
     3781 aaggtcatgt actgggcata atgccaggcg ggccatttac cgtcattgac gtcaataggg
     3841 ggcgtacttg gcatatgata cacttgatgt actgccaagt gggcagttta ccgtaaatac
     3901 tccacccatt gacgtcaatg gaaagtccct attggcgtta ctatgggaac atacgtcatt
     3961 attgacgtca atgggcgggg gtcgttgggc ggtcagccag gcgggccatt taccgtaagt
     4021 tatgtaacgc ggaactccat atatgggcta tgaactaatg accccgtaat tgattactat
     4081 taataactag tcaataatca atgtcgggcg agctcgaatt aattcgtaat catgtcatag
     4141 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     4201 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     4261 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     4321 cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg
     4381 ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg
     4441 ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag
     4501 gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
     4561 gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga
     4621 taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
     4681 accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc
     4741 tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc
     4801 cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta
     4861 agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat
     4921 gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca
     4981 gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
     5041 tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt
     5101 acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct
     5161 cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc
     5221 acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa
     5281 acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
     5341 tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
     5401 ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat
     5461 ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta
     5521 tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt
     5581 aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt
     5641 ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
     5701 ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
     5761 gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc
     5821 gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg
     5881 cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga
     5941 actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta
     6001 ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
     6061 tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
     6121 ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca atattattga
     6181 agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat
     6241 aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc
     6301 attattatca tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg
     6361 cgtttcggtg atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct
     6421 tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc
     6481 gggtgtcggg gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat
     6541 aaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     6601 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagcccgag
     6661 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     6721 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     6781 aaatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     6841 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     6901 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     6961 acacccgccg cgcttaatgc gccgctacag ggcgcgtact atggttgctt tgacgtatgc
     7021 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc cattcgccat
     7081 tcaggctgcg caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc
     7141 tggcgaaagg gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt
     7201 cacgacgttg taaaacgacg gccagtgcca agctgttaac agctttttgc aaaagcctag
     7261 gcctccaaaa aagcctcctc actacttctg gaatagctca gaggccgagg cggcctcggc
     7321 ctctgcataa ataaaaaaaa ttagtcagcc atggggcgga gaatgggcgg aactgggcgg
     7381 agttaggggc gggatgggcg gagttagggg cgggactatg gttgctgact aattgagatg
     7441 catgctttgc atacttctgc ctgctgggga gcctggggac tttccacacc tggttgctga
     7501 ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg actttccaca
     7561 ccctaactga cacacattcc acagctggtt ctttccgcct cagaaggtac ctaaccaagt
     7621 tcctctttca gaggttattt caggccatgg tgctgcgccg aattaattcc cgatccagac
     7681 atgataagat acattgatga gtttggacaa accacaacta gaatgcagtg aaaaaaatgc
     7741 tttatttgtg aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa
     7801 caagttgggc catggcggcc aagcttctgc aggtcgagtt tttttttttt tttttttttt
     7861 tttttttttt catggtccaa gaatgtgc
//
