LOCUS       sequence-312973-        8610 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express Fc-tagged Fzd7/8 subtype NGS Wnt in mammalian cells for 
            organoid research.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8610)
  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 8610)
  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: pEGBacmam-NGS Wnt-Fc
FEATURES             Location/Qualifiers
     source          1..8610
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(join(413..418,419..435,436..441))
                     /gene="intI1 (promoter lies within the coding sequence)"
                     /label=Pc promoter
                     /note="class 1 integron promoter"
     polyA_signal    958..1006
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     1031..1050
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     promoter        complement(1142..1251)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     promoter        1296..1387
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     1312..1329
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     enhancer        1567..1946
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1947..2150
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     2101..2121
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     2147..2171
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     intron          2286..2418
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     primer_bind     2401..2420
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     sig_peptide     2529..2588
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             3483..3503
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4215..4232
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    4268..4856
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4321..4341)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(4739..4750)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    4989..5123
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5026..5045)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5080..5099
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     mobile_element  5152..5317
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7L
                     /note="mini-Tn7 element (left end of the Tn7 transposon)"
     rep_origin      5501..5955
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5588..5607)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5797..5818
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5981..6085
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6086..6154,6155..6946)
                     /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(6304..6323)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7117..7705
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7606..7625
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7859..7876
                     /label=L4440
                     /note="L4440 vector, forward primer"
     mobile_element  8010..8234
                     /mobile_element_type="transposon:Tn7"
                     /label=Tn7R
                     /note="mini-Tn7 element (right end of the Tn7 transposon)"
     CDS             complement(join(8301..8610,1..224))
                     /codon_start=1
                     /gene="aacC1"
                     /product="gentamycin acetyltransferase"
                     /label=GmR
                     /note="confers resistance to gentamycin"
                     /translation="MLRSSNDVTQQGSRPKTKLGGSSMGIIRTCRLGPDQVKSMRAALD
                     LFGREFGDVATYSQHQPDSDYLGNLLRSKTFIALAAFDQEAVVGALAAYVLPKFEQPRS
                     EIYIYDLAVSGEHRRQGIATALINLLKHEANALGAYVIYVQADYGDDPAVALYTKLGIR
                     EEVMHFDIDPSTAT"
ORIGIN
        1 gtcttactac ggagcaagtt cccgaggtaa tcggagtccg gctgatgttg ggagtaggtg
       61 gctacgtctc cgaactcacg accgaaaaga tcaagagcag cccgcatgga tttgacttgg
      121 tcagggccga gcctacatgt gcgaatgatg cccatacttg agccacctaa ctttgtttta
      181 gggcgactgc cctgctgcgt aacatcgttg ctgctgcgta acatcgttgc tgctccataa
      241 catcaaacat cgacccacgg cgtaacgcgc ttgctgcttg gatgcccgag gcatagactg
      301 tacaaaaaaa cagtcataac aagccatgaa aaccgccact gcgccgttac caccgctgcg
      361 ttcggtcaag gttctggacc agttgcgtga tcgcatacgc tacttgcatt acagtttacg
      421 aaccgaacag gcttatgtca actgggttcg tgccttcatc cgtttccacg gtgtgcgtca
      481 cccggcaacc ttgggcagca gcgaagtcga ggcatttctg tcctggctgg cgaacgagcg
      541 caaggtttcg gtctccacgc atcgtcaggc attggcggcc ttgctgttct tctacggcaa
      601 ggtgctgtgc acggatctgc ccttgcttca ggagatcggt agacctcggc cgtcgcggcg
      661 cttgccggtg gtgctgaccc cggatgaagt ggttcgcatc ctcggttttc tggaaggcga
      721 gcatcgtttg ttcgcccagg actctagcta tagttctagt ggttggctac agctttgttt
      781 aaacaaagct ggctatggca gggcttgccg ccccgacgtt ggctgcgagc cctgggcctt
      841 cacccgaact tgggggttgg ggtggggaaa aggaagaaac gcgggcgtat tggtcccaat
      901 ggggtctcgg tggggtatcg acagagtgcc agccctggga ccgaaccccg cgtttatgaa
      961 caaacgaccc aacacccgtg cgttttattc tgtcttttta ttgccgtcat agcgcgggtt
     1021 ccttccggta ttgtctcctt ccgtgtttca gttagcctcc cccatctccc ggtaccgcat
     1081 gctatgcatc agctgctagc accatggctc gagatcccgg gtgatcaagt cttcgtcgag
     1141 tgattgtaaa taaaatgtaa tttacagtat agtattttaa ttaatataca aatgatttga
     1201 taataattct tatttaacta taatatattg tgttgggttg aattaaaggt ccgtatacta
     1261 gtatcgattc gcgacctact ccggaatatt aatagatcat ggagataatt aaaatgataa
     1321 ccatctcgca aataaataag tattttactg ttttcgtaac agttttgtaa taaaaaaacc
     1381 tataaatatt ccggattatt cataccgtcc caccatcggg cgcggatcct caatattggc
     1441 cattagccat attattcatt ggttatatag cataaatcaa tattggctat tggccattgc
     1501 atacgttgta tctatatcat aatatgtaca tttatattgg ctcatgtcca atatgaccgc
     1561 catgttggca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
     1621 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
     1681 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
     1741 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
     1801 tacatcaagt gtatcatatg ccaagtccgc cccctattga cgtcaatgac ggtaaatggc
     1861 ccgcctggca ttatgcccag tacatgacct tacgggactt tcctacttgg cagtacatct
     1921 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacacc aatgggcgtg
     1981 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
     2041 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taataacccc gccccgttga
     2101 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct cgtttagtga
     2161 accgtcagat cactagaagc tttattgcgg tagtttatca cagttaaatt gctaacgcag
     2221 tcagtgcttc tgacacaaca gtctcgaact taagctgcag aagttggtcg tgaggcactg
     2281 ggcaggtaag tatcaaggtt acaagacagg tttaaggaga ccaatagaaa ctgggcttgt
     2341 cgagacagag aagactcttg cgtttctgat aggcacctat tggtcttact gacatccact
     2401 ttgcctttct ctccacaggt gtccactccc agttcaatta cagctcttaa gggatcccgg
     2461 tccgaagcgc gcggaattca aaggcctacg tcgacgagct cactagtcgc ggccgctttc
     2521 gaatcaccat ggagacagac acactcctgc tatgggtact gctgctctgg gttccaggtt
     2581 ccactggtga cgcgagctct gaactgggta cgcgtctgat cagggcagca ttagacggta
     2641 acaaagaccg tgttaaagac ctcattgaaa atggtgctga cgttaacgcg tcccttatgt
     2701 ctggggcgac tccgttacac gccgccgcca tgaacggcca caaagaggtt gtgaagttac
     2761 tgatctccaa gggcgcagat gtgaatgctc agtccgttgc gggttctaca cctctggatg
     2821 cggcggcgtt tagtggacat aaagaagtgg taaaactgct gataagtaaa ggagcagacg
     2881 tcaatgctgt taatgcggct ggattgaccc ccctacatgc tgctgctgat aatgggcaca
     2941 aggaagtagt gaagttgctt atttctaagg gggccgacgt aaatgcgaaa gctgaccatg
     3001 gcatgactcc actccatttc gcagcacagc gcggtcataa ggaagtcgtt aaactattaa
     3061 tcagcaaagg tgcggatttg aacacctctg ccaaagacgg tgcaaccccg cttgacatgg
     3121 cgcgtgaatc tggcaatgag gaggttgtca agctcttgga aaagcaactc gagggaagcg
     3181 gttccggagg ttctggctcc ggaaaaatgt atcacactaa gggacaggaa ggcagcgtgt
     3241 gcctgaggtc ctctgactgt gcatctggcc tgtgctgcgc cagacacttt tggtccaaaa
     3301 tctgcaagcc ggttctgaaa gagggccagg tgtgcacaaa gcatcgacga aagggcagcc
     3361 atggactcga gatctttcag aggtgctact gcggggaggg tctgtcttgc agaatccaga
     3421 aagatcatca tcaggcctcc aactcctccc gcctgcacac ctgccagcga catgcggccg
     3481 cagagaacct gtacttccag ggatcttccg agcccaaatc ttgtgacaaa actcacacat
     3541 gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc ttccccccaa
     3601 aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg gtggtggacg
     3661 tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg gaggtgcata
     3721 atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg gtcagcgtcc
     3781 tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag gtctccaaca
     3841 aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag ccccgagaac
     3901 cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag gtcagcctga
     3961 cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag agcaatgggc
     4021 agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc tccttcttcc
     4081 tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc ttctcatgct
     4141 ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctcc ctgtctccgg
     4201 gtaaaggggc cgcacatcac catcaccatc actaagctcc gctagagcct gcagtctcga
     4261 caagcttaat caacctctgg attacaaaat ttgtgaaaga ttgactggta ttcttaacta
     4321 tgttgctcct tttacgctat gtggatacgc tgctttaatg cctttgtatc atgctattgc
     4381 ttcccgtatg gctttcattt tctcctcctt gtataaatcc tggttgctgt ctctttatga
     4441 ggagttgtgg cccgttgtca ggcaacgtgg cgtggtgtgc actgtgtttg ctgacgcaac
     4501 ccccactggt tggggcattg ccaccacctg tcagctcctt tccgggactt tcgctttccc
     4561 cctccctatt gccacggcgg aactcatcgc cgcctgcctt gcccgctgct ggacaggggc
     4621 tcggctgttg ggcactgaca attccgtggt gttgtcgggg aagctgacgt cctttccatg
     4681 gctgctcgcc tgtgttgcca cctggattct gcgcgggacg tccttctgct acgtcccttc
     4741 ggccctcaat ccagcggacc ttccttcccg cggcctgctg ccggctctgc ggcctcttcc
     4801 gcgtcttcgc cttcgccctc agacgagtcg gatctccctt tgggccgcct ccccgcctga
     4861 agcttgtcga gaagtactag aggatcataa tcagccatac cacatttgta gaggttttac
     4921 ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg
     4981 ttgttgttaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     5041 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     5101 atgtatctta tcatgtctgg atctgatcac tgcttgagcc taggagatcc gaaccagata
     5161 agtgaaatct agttccaaac tattttgtca tttttaattt tcgtattagc ttacgacgct
     5221 acacccagtt cccatctatt ttgtcactct tccctaaata atccttaaaa actccatttc
     5281 cacccctccc agttcccaac tattttgtcc gcccacagcg gggcattttt cttcctgtta
     5341 tgtttttaat caaacatcct gccaactcca tgtgacaaac cgtcatcttc ggctactttt
     5401 tctctgtcac agaatgaaaa tttttctgtc atctcttcgt tattaatgtt tgtaattgac
     5461 tgaatatcaa cgcttatttg cagcctgaat ggcgaatggg acgcgccctg tagcggcgca
     5521 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     5581 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     5641 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     5701 cccaaaaaac ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt
     5761 tttcgccctt tgacgttgga gtccacgttc ttaatagtgg actcttgttc caaactggaa
     5821 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     5881 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     5941 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     6001 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct
     6061 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
     6121 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
     6181 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
     6241 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
     6301 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
     6361 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
     6421 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
     6481 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
     6541 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
     6601 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
     6661 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
     6721 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
     6781 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
     6841 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     6901 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     6961 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     7021 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     7081 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     7141 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     7201 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     7261 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     7321 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     7381 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     7441 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     7501 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     7561 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     7621 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     7681 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     7741 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     7801 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     7861 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc ttacgcatct
     7921 gtgcggtatt tcacaccgca tagaccagcc gcgtaacctg gcaaaatcgg ttacggttga
     7981 gtaataaatg gatgccctgc gtaagcgggt gtgggcggac aataaagtct taaactgaac
     8041 aaaatagatc taaactatga caataaagtc ttaaactaga cagaatagtt gtaaactgaa
     8101 atcagtccag ttatgctgtg aaaaagcata ctggactttt gttatggcta aagcaaactc
     8161 ttcattttct gaagtgcaaa ttgcccgtcg tattaaagag gggcgtggcc aagggcatgg
     8221 taaagactat attcgcggcg ttgtgacaat ttaccgaaca actccgcggc cgggaagccg
     8281 atctcggctt gaacgaattg ttaggtggcg gtacttgggt cgatatcaaa gtgcatcact
     8341 tcttcccgta tgcccaactt tgtatagaga gccactgcgg gatcgtcacc gtaatctgct
     8401 tgcacgtaga tcacataagc accaagcgcg ttggcctcat gcttgaggag attgatgagc
     8461 gcggtggcaa tgccctgcct ccggtgctcg ccggagactg cgagatcata gatatagatc
     8521 tcactacgcg gctgctcaaa cttgggcaga acgtaagccg cgagagcgcc aacaaccgct
     8581 tcttggtcga aggcagcaag cgcgatgaat
//
