LOCUS       sequence-224035-        7329 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses and secretes human Norrin with N-terminal flag and 
            alkaline phosphatase tags, contains artificial signal sequence 
            upstream of tags..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7329)
  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 7329)
  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"; 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: pcDNA3.3 ss-FLAG-AP-hNDP (aa 25-133)
FEATURES             Location/Qualifiers
     source          1..7329
                     /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..848
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2715..2734)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    2759..2807
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      3009..3437
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3096..3115)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3306..3327
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3446..3466)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3451..3780
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3631..3766
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3693..3712
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3847..4641
                     /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(3901..3920)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4511..4530
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4817..4938
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4854..4873)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4908..4927
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4987..5003)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4987..5003)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5000..5022)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5011..5027
                     /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(5035..5041,5042..5059,5060..5065))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5080..5101
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5218..5235)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5389..5977)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5469..5488)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6148..6939,6940..7008))
                     /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     6771..6790
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7009..7113)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7188..7207)
                     /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 caaagactac aaggacgacg
      841 atgacaaggc ggccgccatc atcccagttg aggaggagaa cccggacttc tggaaccgcg
      901 aggcagccga ggccctgggt gccgccaaga agctgcagcc tgcacagaca gccgccaaga
      961 acctcatcat cttcctgggc gatgggatgg gggtgtctac ggtgacagct gccaggatcc
     1021 taaaagggca gaagaaggac aaactggggc ctgagatacc cctggccatg gaccgcttcc
     1081 catatgtggc tctgtccaag acatacaatg tagacaaaca tgtgccagac agtggagcca
     1141 cagccacggc ctacctgtgc ggggtcaagg gcaacttcca gaccattggc ttgagtgcag
     1201 ccgcccgctt taaccagtgc aacacgacac gcggcaacga ggtcatctcc gtgatgaatc
     1261 gggccaagaa agcagggaag tcagtgggag tggtaaccac cacacgagtg cagcacgcct
     1321 cgccagccgg cacctacgcc cacacggtga accgcaactg gtactcggac gccgacgtgc
     1381 ctgcctcggc ccgccaggag gggtgccagg acatcgctac gcagctcatc tccaacatgg
     1441 acattgacgt gatcctaggt ggaggccgaa agtacatgtt tcgcatggga accccagacc
     1501 ctgagtaccc agatgactac agccaaggtg ggaccaggct ggacgggaag aatctggtgc
     1561 aggaatggct ggcgaagcgc cagggtgccc ggtatgtgtg gaaccgcact gagctcatgc
     1621 aggcttccct ggacccgtct gtgacccatc tcatgggtct ctttgagcct ggagacatga
     1681 aatacgagat ccaccgagac tccacactgg acccctccct gatggagatg acagaggctg
     1741 ccctgcgcct gctgagcagg aacccccgcg gcttcttcct cttcgtggag ggtggtcgca
     1801 tcgaccatgg tcatcatgaa agcagggctt accgggcact gactgagacg atcatgttcg
     1861 acgacgccat tgagagggcg ggccagctca ccagcgagga ggacacgctg agcctcgtca
     1921 ctgccgacca ctcccacgtc ttctccttcg gaggctaccc cctgcgaggg agctccatct
     1981 tcgggctggc ccctggcaag gcccgggaca ggaaggccta cacggtcctc ctatacggaa
     2041 acggtccagg ctatgtgctc aaggacggcg cccggccgga tgttaccgag agcgagagcg
     2101 ggagccccga gtatcggcag cagtcagcag tgcccctgga cgaagagacc cacgcaggcg
     2161 aggacgtggc ggtgttcgcg cgcggcccgc aggcgcacct ggttcacggc gtgcaggagc
     2221 agaccttcat agcgcacgtc atggccttcg ccgcctgcct ggagccctac accgcctgcg
     2281 acctggcgcc ccccgccggc accaccgacg ccgcgcaccc gggttatctc gagaaaacgg
     2341 acagctcatt cataatggac tcggaccctc gacgctgcat gaggcaccac tatgtggatt
     2401 ctatcagtca cccattgtac aagtgtagct caaagatggt gctcctggcc aggtgcgagg
     2461 ggcactgcag ccaggcgtca cgctccgagc ctttggtgtc gttcagcact gtcctcaagc
     2521 aacccttccg ttcctcctgt cactgctgcc ggccccagac ttccaagctg aaggcactgc
     2581 ggctgcgatg ctcagggggc atgcgactca ctgccaccta ccggtacatc ctctcctgtc
     2641 actgcgagga atgcaattcc tgaaagggtt cgatccctac cggttagtaa tgagtttaaa
     2701 cgggggaggc taactgaaac acggaaggag acaataccgg aaggaacccg cgctatgacg
     2761 gcaataaaaa gacagaataa aacgcacggg tgttgggtcg tttgttcata aacgcggggt
     2821 tcggtcccag ggctggcact ctgtcgatac cccaccgaga ccccattggg gccaatacgc
     2881 ccgcgtttct tccttttccc caccccaccc cccaagttcg ggtgaaggcc cagggctcgc
     2941 agccaacgtc ggggcggcag gccctgccat agcagatctg cgcagctggg gctctagggg
     3001 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     3061 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     3121 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     3181 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3241 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3301 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3361 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3421 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3481 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3541 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3601 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3661 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     3721 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3781 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     3841 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     3901 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     3961 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     4021 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     4081 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     4141 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     4201 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4261 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4321 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     4381 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     4441 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     4501 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     4561 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     4621 cttcttgacg agttcttctg agcgggactc tggggttcgc gaaatgaccg accaagcgac
     4681 gcccaacctg ccatcacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     4741 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     4801 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     4861 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     4921 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     4981 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     5041 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     5101 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     5161 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     5221 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     5281 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5341 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5401 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5461 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5521 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5581 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5641 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5701 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5761 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5821 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5881 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5941 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     6001 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     6061 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     6121 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     6181 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     6241 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6301 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6361 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6421 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6481 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6541 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6601 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6661 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6721 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6781 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6841 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6901 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6961 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     7021 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     7081 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga
     7141 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
     7201 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
     7261 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
     7321 ctgcttagg
//
