LOCUS       sequence-360377-        6956 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses FLAG tagged FZD4 swapped with FZD5 CRD.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6956)
  AUTHORS   Ko SB, Mihara E, Park Y, Roh K, Kang C, Takagi J, Bang I, Choi HJ
  TITLE     Functional role of the Frizzled linker domain in the Wnt signaling 
            pathway.
  JOURNAL   Commun Biol. 2022 May 5;5(1):421. doi: 10.1038/s42003-022-03370-4.
  PUBMED    35513706
REFERENCE   2  (bases 1 to 6956)
  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: "Commun 
            Biol."; date: "2022-05-5"; volume: "5(1):421. doi"; pages: " 
            10.1038/s42003-022-03370-4"
COMMENT     Sequence Label: pcDNA FZD4_5CRD
FEATURES             Location/Qualifiers
     source          1..6956
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     9..30
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(149..169)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        154..483
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      334..469
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     396..415
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             550..1344
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(604..623)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1214..1233
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    1520..1641
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1557..1576)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1611..1630
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1690..1706)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1690..1706)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1703..1725)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1714..1730
                     /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(1738..1744,1745..1762,1763..1768))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1783..1804
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1921..1938)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2092..2680)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2172..2191)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2851..3642,3643..3711))
                     /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     3474..3493
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3712..3816)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(3891..3910)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4082..4461
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4462..4665
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4616..4636
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4710..4729
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4710..4728
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             4762..4806
                     /codon_start=1
                     /product="leader sequence from mouse preprotrypsin"
                     /label=preprotrypsin leader
                     /translation="MSALLILALVGAAVA"
     CDS             4807..4830
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             6352..6369
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6392..6409)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6398..6622
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      join(6668..6956,1..140)
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6755..6774)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
       61 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
      121 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
      181 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
      241 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
      301 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
      361 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
      421 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
      481 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga
      541 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
      601 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
      661 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
      721 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
      781 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
      841 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
      901 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
      961 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     1021 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     1081 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     1141 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     1201 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     1261 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     1321 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgcgaaatga ccgaccaagc
     1381 gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg aaaggttggg
     1441 cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct
     1501 ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa
     1561 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     1621 caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc
     1681 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     1741 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     1801 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     1861 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     1921 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     1981 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2041 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2101 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     2161 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     2221 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     2281 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     2341 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     2401 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     2461 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     2521 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     2581 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     2641 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     2701 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     2761 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     2821 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     2881 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     2941 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     3001 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     3061 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     3121 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     3181 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     3241 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     3301 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     3361 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     3421 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     3481 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     3541 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     3601 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     3661 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     3721 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     3781 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     3841 tgacgtcgac ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc
     3901 tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag
     3961 tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag
     4021 aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag atatacgcgt
     4081 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
     4141 ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
     4201 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
     4261 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat
     4321 caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
     4381 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
     4441 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag
     4501 cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
     4561 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
     4621 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctctctg gctaactaga
     4681 gaacccactg cttactggct tatcgaaatt aatacgactc actataggga gacccaagct
     4741 ggctagttaa gcttggtacc aatgtctgca cttctgatcc tagctcttgt tggagctgca
     4801 gttgctgact acaaagacga tgacgacaag cttgcggccg cagcgtccaa ggccccggtg
     4861 tgccaggaaa tcacggtgcc catgtgccgc ggcatcggct acaacctgac gcacatgccc
     4921 aaccagttca accacgacac gcaggacgag gcgggcctgg aggtgcacca gttctggccg
     4981 ctggtggaga tccaatgctc gccggacctg cgcttcttcc tatgctctat gtacacgccc
     5041 atctgtctgc ccgactacca caagccgctg ccgccctgcc gctcggtgtg cgagcgcgcc
     5101 aaggccggct gctcgccgct gatgcgccag tacggcttcg cctggcccga gcgcatgagc
     5161 tgcgaccgcc tcccggtgct gggccgcgac gccgaggtcc tctgcatgga tggaccaggt
     5221 gatgaagagg ttcccttgcc ccacaagact cccatccagc ccggggaaga gtgccactcc
     5281 gtgggaagca attctgatca gtacatctgg gtgaagagga gcctgaactg tgttctcaag
     5341 tgtggctacg atgctggctt gtacagccgc tcagctaagg agttcacgga tatttggatg
     5401 gctgtgtggg ccagcctctg cttcatctcc accaccttca ccgtgctgac cttcctgatt
     5461 gattcatcca ggttttctta ccctgagcgc cccatcatat ttctcagtat gtgctataat
     5521 atttatagca ttgcttatat tgttcggctg actgtaggcc gggaaaggat atcctgtgat
     5581 tttgaagagg cggcagagcc cgttctcatc caagaaggac ttaagaacac aggatgtgca
     5641 ataattttct tgctgatgta cttttttgga atggccagct ccatttggtg ggttattctg
     5701 acactcactt ggtttttggc agccggactc aagtggggtc atgaagccat tgaaatgcac
     5761 agttcttatt tccacatcgc agcctgggct attcccgcag tgaaaaccat tgtcatcttg
     5821 attatgagac tagtggatgc cgatgaactg actggcttgt gctatgttgg gaaccaaaac
     5881 ctagatgccc tcactggctt tgtggtggct cctctcttta cgtatttggt gattggaacg
     5941 ctgttcattg cggcgggttt ggtggcctta ttcaaaattc ggtccaatct tcaaaaagac
     6001 gggacaaaga cagacaagtt ggaaaggcta atggtcaaga tcggggtctt ctcagtactg
     6061 tacacggttc ctgcaacctg tgtgattgcc tgttatttct atgaaatctc aaactgggca
     6121 ctctttcgat attctgcaga tgactcaaac atggcagttg aaatgttgaa aatttttatg
     6181 tctttgctcg tgggcatcac ttcaggcatg tggatttggt ctgccaaaac tcttcacacg
     6241 tggcaaaagt gttctaaccg attggtgaat tctgggaagg taaagagaga gaagaggggg
     6301 aatggttggg tgaagccagg aaaaggcaac gagactgtgg tatagctcga gcatcatcac
     6361 catcaccatt gagtttaaac ccgctgatca gcctcgactg tgccttctag ttgccagcca
     6421 tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc
     6481 ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg
     6541 gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct
     6601 ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg ctctaggggg
     6661 tatccccacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     6721 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     6781 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     6841 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     6901 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgtt
//
