LOCUS       sequence-313516-        8470 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Fzd8 subtype NGS Wnt in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8470)
  AUTHORS   Miao Y, Ha A, de Lau W, Yuki K, Santos AJM, You C, Geurts MH, 
            Puschhof J, Pleguezuelos-Manzano C, Peng WC, Senlice R, Piani C, 
            Buikema JW, Gbenedio OM, Vallon M, Yuan J, de Haan S, Hemrika W, 
            Rosch K, Dang LT, Baker D, Ott M, Depeille P, Wu SM, Drost J, Nusse 
            R, Roose JP, Piehler J, Boj SF, Janda CY, Clevers H, Kuo CJ, Garcia 
            KC
  TITLE     Next-Generation Surrogate Wnts Support Organoid Growth and 
            Deconvolute Frizzled Pleiotropy In Vivo.
  JOURNAL   Cell Stem Cell. 2020 Aug 14. pii: S1934-5909(20)30358-1. doi: 
            10.1016/j.stem.2020.07.020.
  PUBMED    32818433
REFERENCE   2  (bases 1 to 8470)
  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: "Cell Stem 
            Cell. 2020 Aug 14. pii: S1934-5909(20)30358-1. doi: 
            10.1016/j.stem.2020.07.020."
COMMENT     Sequence Label: RKS-Fzd8 subtype NGS Wnt
FEATURES             Location/Qualifiers
     source          1..8470
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..227
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             228..408
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    455..580
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1072..1305
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1490..1534
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    1801..1918
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2615..3016
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     CDS             3972..3995
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     misc_feature    4015..4582
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(4176..4193)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4403..4422
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     regulatory      4605..4614
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4611..4613,4614..4616,4617..5330)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(4611..4613,4614..4616,4617..5327)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4656..4677)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4917..4936)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     5264..5285
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(6001..6034)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(6239..6256)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6245..6469
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6542..6559)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6713..7301)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6793..6812)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7472..8263,8264..8332))
                     /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     8095..8114
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8333..8437)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 tgtagtctta tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg
       61 ccttacaagg agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc
      121 gtgccttatt aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc
      181 cgcattgcag agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt
      241 tagaccagat ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc
      301 aataaagctt gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta
      361 actagagatc cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa
      421 cagggacttg aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc
      481 tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac
      541 tagcggaggc tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat
      601 tagatcgcga tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa
      661 acatatagta tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga
      721 aacatcagaa ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc
      781 agaagaactt agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat
      841 agagataaaa gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa
      901 gaccaccgca cagcaagcgg ccctgatctt cagacctgga ggaggagata tgagggacaa
      961 ttggagaagt gaattatata aatataaagt agtaaaaatt gaaccattag gagtagcacc
     1021 caccaaggca aagagaagag tggtgcagag agaaaaaaga gcagtgggaa taggagcttt
     1081 gttccttggg ttcttgggag cagcaggaag cactatgggc gcagcgtcaa tgacgctgac
     1141 ggtacaggcc agacaattat tgtctggtat agtgcagcag cagaacaatt tgctgagggc
     1201 tattgaggcg caacagcatc tgttgcaact cacagtctgg ggcatcaagc agctccaggc
     1261 aagaatcctg gctgtggaaa gatacctaaa ggatcaacag ctcctgggga tttggggttg
     1321 ctctggaaaa ctcatttgca ccactgctgt gccttggaat gctagttgga gtaatgaatc
     1381 tctggaacag atttggaatc acacgacctg gatggagtgg gacagagaaa ttaacaatta
     1441 cacaagctta atacactcct taattgaaga atcgcaaaac cagcaagaaa agaatgaaca
     1501 agaattattg gaattagata aatgggcaag tttgtggaat tggtttaaca taacaaattg
     1561 gctgtggtat ataaaattat tcataatgat agtaggaggc ttggtaggtt taagaatagt
     1621 ttttgctgta ctttctatag tgaatagagt taggcaggga tattcaccat tatcgtttca
     1681 gacccacctc ccaaccccga ggggacccga caggcccgaa ggaatagaag aagaaggtgg
     1741 agagagagac agagacagat ccattcgatt agtgaacgga tctcgacggt atcggttaac
     1801 ttttaaaaga aaagggggga ttggggggta cagtgcaggg gaaagaatag tagacataat
     1861 agcaacagac atacaaacta aagaattaca aaaacaaatt acaaaaattc aaaattttat
     1921 cgattacgcg tcactggcat ctgaagcacc accagcgagc gagagctaga gagaaggaaa
     1981 gccaccgact tcaccgcctc cgagctgctc cgggtcgcgg gtctgcagcg tctccggccc
     2041 tccgcgccta cagctcaagc cacatccgaa gggggaggga gccgggagct gcgcgcgggg
     2101 ccgctggggg gaggggtggc accgcccacg ccgggcggcc acgaagggcg gggcagcggg
     2161 cgcgcgcccg gcggggggag gggccgcgcg ccgcgcccgc tgggaattgg ggccctaggg
     2221 ggagggcgga ggcgccgacg accgcggcac ttaccgttcg cggcgtggcg cccggtggtc
     2281 cccaagggga gggaaggggg aggcggggcg aggacagtga ccggagtctc ctcagcggtg
     2341 gcttttctgc ttggcagcct cagcggctgg cgccaaaacc ggactccgcc cacttcctcg
     2401 cccctgcggt gcgagggtgt ggaatcctcc agacgctggg ggagggggag ttgggagctt
     2461 aaaaactagt acccctttgg gaccactttc agcagcgaac tctcctgtac accaggggtc
     2521 agttccacag acgcgggcca ggggtgggtc attgcggcgt gaacaataat ttgactagaa
     2581 gttgattcgg gtgtttccgg aattcctagc tgcagtaacg ccattttgca aggcatggaa
     2641 aaataccaaa ccaagaatag agaagttcag atcaagggcg ggtacatgaa aatagctaac
     2701 gttgggccaa acaggatatc tgcggtgagc agtttcggcc ccggcccggg gccaagaaca
     2761 gatggtcacc gcagtttcgg ccccggcccg aggccaagaa cagatggtcc ccagatatgg
     2821 cccaaccctc agcagtttct taagacccat cagatgtttc caggctcccc caaggacctg
     2881 aaatgaccct gcgccttatt tgaattaacc aatcagcctg cttctcgctt ctgttcgcgc
     2941 gcttctgctt cccgagctct ataaaagagc tcacaacccc tcactcggcg cgccagtcct
     3001 ccgacagact gagtcgcccg ctcgaggcta gcatgaagac catcatcgcc ctgagctaca
     3061 tcttctgcct ggtgttcgcc ggatcctctg aactgggtaa acgtctgatc atggcagcat
     3121 tggacggtaa caaagaccgt gttaaagacc tcattgaaaa tggtgctgac gttaacgcgt
     3181 ccctcgtgtc tggggcgact ccgttacacg ccgccgccat gaacggccac aaagaggttg
     3241 tgaagttact gatctccaag ggcgcagatg tgaatgctca gtccgcggcg ggttctacac
     3301 ctctggctgc ggcggcgatc aatggacata aagaagtggt aaaactgctg ataagtaaag
     3361 gagcagacgt caatgctgtt acggcggctg gaatgacccc cctacatgct gctgctgcta
     3421 atgggcacaa ggaagtagtg aagttgctta tttctaaggg ggccgacgta aatgcgaaag
     3481 cggaccgtgg catgactcca ctccatttcg cagcatggcg cggtcataag gaagtcgtta
     3541 aactattaat cagcaaaggt gcggatttga acacctctgc caaagacggt gcaaccccgc
     3601 ttgacatggc gcgtgaatct ggcaatgagg aggttgtcaa gctcttggaa aagcaactcg
     3661 agggcagcgg ctccggaggc agcggctccg gaaaaatgta tcacactaag ggacaggaag
     3721 gcagcgtgtg cctgaggtcc tctgactgtg catctggcct gtgctgcgcc agacactttt
     3781 ggtccaaaat ctgcaagccg gttctgaaag agggccaggt gtgcacaaag catcgacgaa
     3841 agggcagcca tggactcgag atctttcaga ggtgctactg cggggagggt ctgtcttgca
     3901 gaatccagaa agatcatcat caggcctcca actcctcccg cctgcacacc tgccagcgac
     3961 atgcggccgc gcatcatcac caccatcacc accattaaga tcccggaccg cctctccctc
     4021 ccccccccct aacgttactg gccgaagccg cttggaataa ggccggtgtg cgtttgtcta
     4081 tatgttattt tccaccatat tgccgtcttt tggcaatgtg agggcccgga aacctggccc
     4141 tgtcttcttg acgagcattc ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct
     4201 gttgaatgtc gtgaaggaag cagttcctct ggaagcttct tgaagacaaa caacgtctgt
     4261 agcgaccctt tgcaggcagc ggaacccccc acctggcgac aggtgcctct gcggccaaaa
     4321 gccacgtgta taagatacac ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg
     4381 gatagttgtg gaaagagtca aatggctctc ctcaagcgta ttcaacaagg ggctgaagga
     4441 tgcccagaag gtaccccatt gtatgggatc tgatctgggg cctcggtgca catgctttac
     4501 atgtgtttag tcgaggttaa aaaaacgtct aggccccccg aaccacgggg acgtggtttt
     4561 cctttgaaaa acacgatgat aagcttgcca caacccttat cgaggccacc atggtgagca
     4621 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     4681 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     4741 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     4801 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     4861 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     4921 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     4981 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     5041 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     5101 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     5161 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     5221 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     5281 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtga atctagagtc
     5341 gaccgagcat cttaccgcca tttataccca tatttgttct gtttttcttg atttgggtat
     5401 acatttaaat gttaatagaa caaaatggtg gggcaatcat ttacattttt agggatatgt
     5461 aattactagt tcaggtgtat tgccacaaga caaacatgtt aagaaacttt cccgttattt
     5521 acgctctgtt cctgttaatc aacctctgga ttacaaaatt tgtgaaagat tgactgatat
     5581 tcttaactat gttgctcctt ttacgctgtg tggatatgct gctttatagc ctctgtatct
     5641 agctattgct tcccgtacgg ctttcgtttt ctcctccttg tataaatcct ggttgctgtc
     5701 tcttttagag gagttgtggc ccgttgtccg tcaacgtggc gtggtgtgct ctgtgtttgc
     5761 tgacgcaacc cccactggct ggggcattgc caccacctgt caactccttt ctgggacttt
     5821 cgctttcccc ctcccgatcg ccacggcaga actcatcgcc gcctgccttg cccgctgctg
     5881 gacaggggct aggttgctgg gcactgataa ttccgtggtg ttgtcatcga attcggtacc
     5941 tttttaaaag aaaagggggg actggaaggg ctaattcact cccaacgaag acaagatatc
     6001 ataacttcgt atagcataca ttatacgaag ttataattta tttgtgaaat ttgtgatgct
     6061 attgctttat ttgtaaccat atgtttattt gtgaaatttg tgatgctatt gctttatttg
     6121 taaccattgc tttttgcttg tactgggtct ctctggttag accagatctg agcctgggag
     6181 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc tcgaccagcc
     6241 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     6301 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     6361 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     6421 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc ctgcagctgc
     6481 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     6541 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     6601 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     6661 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     6721 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     6781 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     6841 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     6901 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     6961 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     7021 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     7081 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     7141 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     7201 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     7261 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     7321 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     7381 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     7441 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     7501 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     7561 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     7621 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     7681 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     7741 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     7801 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     7861 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     7921 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     7981 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     8041 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     8101 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     8161 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     8221 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     8281 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     8341 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     8401 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     8461 ctgacgtcaa
//
