LOCUS       sequence-218824-        7047 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses human FZD4 with N-terminal V5-tag in mammalian cells, 
            contains artificial signal sequence upstream of tag..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7047)
  AUTHORS   Lai MB, Zhang C, Shi J, Johnson V, Khandan L, McVey J, Klymkowsky 
            MW, Chen Z, Junge HJ
  TITLE     TSPAN12 Is a Norrin Co-receptor that Amplifies Frizzled4 Ligand 
            Selectivity and Signaling.
  JOURNAL   Cell Rep. 2017 Jun 27;19(13):2809-2822. doi: 
            10.1016/j.celrep.2017.06.004.
  PUBMED    28658627
REFERENCE   2  (bases 1 to 7047)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.celrep.2017.06"; journalName: "Cell Rep"; date: 
            "2017-06-27- 27"; volume: "19"; issue: "13"; pages: "2809-2822"
COMMENT     Sequence Label: pcDNA 3.3 ss-V5-hFZD4 (aa 37-537)
FEATURES             Location/Qualifiers
     source          1..7047
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        50..429
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        430..633
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     584..604
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     630..654
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             825..866
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2433..2452)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2477..2525
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2727..3155
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2814..2833)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3024..3045
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3164..3184)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3169..3498
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3349..3484
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3411..3430
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3565..4359
                     /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(3619..3638)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4229..4248
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4535..4656
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4572..4591)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4626..4645
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4705..4721)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4705..4721)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4718..4740)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4729..4745
                     /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(4753..4759,4760..4777,4778..4783))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4798..4819
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4936..4953)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5107..5695)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5187..5206)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5866..6657,6658..6726))
                     /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     6489..6508
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6727..6831)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6906..6925)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttaggcgtt ttgcgctgct tcgcgatgta cgggccagat atacgcgttg acattgatta
       61 ttgactagtt attaatagta atcaattacg gggtcattag ttcatagccc atatatggag
      121 ttccgcgtta cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc
      181 ccattgacgt caataatgac gtatgttccc atagtaacgc caatagggac tttccattga
      241 cgtcaatggg tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat
      301 atgccaagta cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc
      361 cagtacatga ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct
      421 attaccatgg tgatgcggtt ttggcagtac atcaatgggc gtggatagcg gtttgactca
      481 cggggatttc caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat
      541 caacgggact ttccaaaatg tcgtaacaac tccgccccat tgacgcaaat gggcggtagg
      601 cgtgtacggt gggaggtcta tataagcaga gctcgtttag tgaaccgtca gatcgcctgg
      661 agacgccatc cacgctgttt tgacctccat agaagacacc gggaccgatc cagcctccgg
      721 actctagagg atcgaaccct tccaccatgg gagggactgc cgccaggttg ggggccgtga
      781 ttttgtttgt cgtcatagtg ggcctccatg gggtccgcgg caaaggtaag cctatcccta
      841 accctctcct cggtctcgat tctacggcgg ccgccttcgg ggacgaggaa gagcggcgct
      901 gcgaccccat ccgcatctcc atgtgccaga acctcggcta caacgtgacc aagatgccca
      961 acctggttgg gcacgagctg cagacggacg ccgagctgca gctgacaact ttcacaccgc
     1021 tcatccagta cggctgctcc agccagctgc agttcttcct ttgttctgtt tatgtgccaa
     1081 tgtgcacaga gaagatcaac atccccattg gcccatgcgg cggcatgtgt ctttcagtca
     1141 agagacgctg tgaacccgtc ctgaaggaat ttggatttgc ctggccagag agtctgaact
     1201 gcagcaaatt cccaccacag aacgaccaca accacatgtg catggaaggg ccaggtgatg
     1261 aagaggtgcc cttacctcac aaaaccccca tccagcctgg ggaagagtgt cactctgtgg
     1321 gaaccaattc tgatcagtac atctgggtga aaaggagcct gaactgtgtg ctcaagtgtg
     1381 gctatgatgc tggcttatac agccgctcag ccaaggagtt cactgatatc tggatggctg
     1441 tgtgggccag cctgtgtttc atctccactg ccttcacagt actgaccttc ctgatcgatt
     1501 cttctaggtt ttcctaccct gagcgcccca tcatatttct cagtatgtgc tataatattt
     1561 atagcattgc ttatattgtc aggctgactg taggccggga aaggatatcc tgtgattttg
     1621 aagaggcagc agaacctgtt ctcatccaag aaggacttaa gaacacagga tgtgcaataa
     1681 ttttcttgct gatgtacttt tttggaatgg ccagctccat ttggtgggtt attctgacac
     1741 tcacttggtt tttggcagca ggactcaaat ggggtcatga agccattgaa atgcacagct
     1801 cttatttcca cattgcagcc tgggccatcc ccgcagtgaa aaccattgtc atcttgatta
     1861 tgagactggt ggatgcagat gaactgactg gcttgtgcta tgttggaaac caaaatctcg
     1921 atgccctcac cgggttcgtg gtggctcccc tctttactta tttggtcatt ggaactttgt
     1981 tcattgctgc aggtttggtg gccttgttca aaattcggtc aaatcttcaa aaggatggga
     2041 caaagacaga caagttagaa agactgatgg tcaagattgg ggtgttctca gtactgtaca
     2101 cagttcctgc aacgtgtgtg attgcctgtt atttttatga aatctccaac tgggcacttt
     2161 ttcggtattc tgcagatgat tccaacatgg ctgttgaaat gttgaaaatt tttatgtctt
     2221 tgttggtggg catcacttca ggcatgtgga tttggtctgc caaaactctt cacacgtggc
     2281 agaagtgttc caacagattg gtgaattctg gaaaggtaaa gagagagaag agaggaaatg
     2341 gttgggtgaa gcctggaaaa ggcagtgaga ctgtggtata aaagggttcg atccctaccg
     2401 gttagtaatg agtttaaacg ggggaggcta actgaaacac ggaaggagac aataccggaa
     2461 ggaacccgcg ctatgacggc aataaaaaga cagaataaaa cgcacgggtg ttgggtcgtt
     2521 tgttcataaa cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc
     2581 ccattggggc caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg
     2641 tgaaggccca gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cagatctgcg
     2701 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     2761 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2821 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2881 gggcatccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2941 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3001 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3061 tatctcggtc tattcttttg atttataagg gattttgggg atttcggcct attggttaaa
     3121 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     3181 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3241 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3301 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3361 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3421 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3481 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     3541 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     3601 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     3661 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     3721 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     3781 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     3841 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     3901 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     3961 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4021 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4081 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4141 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4201 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4261 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4321 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgcga
     4381 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     4441 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     4501 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     4561 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4621 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     4681 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     4741 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     4801 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     4861 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     4921 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     4981 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     5041 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     5101 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     5161 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     5221 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     5281 gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     5341 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     5401 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     5461 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     5521 gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc
     5581 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     5641 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     5701 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     5761 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     5821 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     5881 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     5941 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     6001 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     6061 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     6121 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     6181 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     6241 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     6301 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     6361 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     6421 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     6481 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     6541 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     6601 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     6661 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     6721 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     6781 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     6841 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtcgact
     6901 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
     6961 ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc
     7021 gacaattgca tgaagaatct gcttagg
//
