LOCUS       sequence-218715-        6377 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human TSPAN12 with N-terminal HA-tag in mammalian cells. 
            Contains multiple silent mutations that introduce restriction sites.
            .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6377)
  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 6377)
  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: pcDNA 3.3 HA-hTSPAN12
FEATURES             Location/Qualifiers
     source          1..6377
                     /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             758..784
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     primer_bind     complement(1763..1782)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    1807..1855
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      2057..2485
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2144..2163)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2354..2375
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2494..2514)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2499..2828
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2679..2814
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2741..2760
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2895..3689
                     /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(2949..2968)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3559..3578
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3865..3986
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3902..3921)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3956..3975
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4035..4051)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4035..4051)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4048..4070)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4059..4075
                     /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(4083..4089,4090..4107,4108..4113))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4128..4149
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4266..4283)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4437..5025)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4517..4536)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5196..5987,5988..6056))
                     /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     5819..5838
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6057..6161)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6236..6255)
                     /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 tggcgcgccc caccatgtac ccatacgatg ttccagatta
      781 cgctctcgag gccagagaag attccgtgaa gtgtctgcgc tgcctgctct acgccctcaa
      841 tctgctcttt tggttaatgt ccatcagtgt gttggcagtt tctgcttgga tgagggacta
      901 cctaaataat gttctcactt taactgcaga aacgagggta gaggaagcag tcattttgac
      961 ttactttcct gtggttcatc cggtcatgat tgctgtttgc tgtttcctta tcattgtggg
     1021 gatgttagga tattgtggaa cggtgaaaag aaatctgttg cttcttgcat ggtactttgg
     1081 aagcttgctt gtcattttct gtgtagaact ggcttgtggc gtttggacat atgaacagga
     1141 acttatggta ccagtacaat ggtcagatat ggtcactttg aaagccagga tgacaaatta
     1201 tggattacct agatatcgat ggcttactca tgcttggaat ttttttcaga gagagtttaa
     1261 gtgctgtgga gtagtatatt tcactgactg gttggaaatg acagagatgg actggccccc
     1321 agattcctgc tgtgttagag aattcccagg atgttccaaa caggcccacc aggaagatct
     1381 cagtgacctt tatcaagagg gttgtgggaa gaaaatgtat tcctttttga gaggaaccaa
     1441 acaactgcag gtgctgaggt ttctgggaat ctccattggg gtgacccaaa tcctggccat
     1501 gattctcacc attactctgc tctgggccct gtattatgat agaagggagc cggggacaga
     1561 ccaaatgatg tccttgaaga atgacaactc tcagcacctg tcatgtccct cagtagaact
     1621 gttgaaacca agcctgtcaa gaatctttga acacacatcc atggcaaaca gctttaatac
     1681 acactttgag atggaggagt tataagctag caagggttcg atccctaccg gttagtaatg
     1741 agtttaaacg ggggaggcta actgaaacac ggaaggagac aataccggaa ggaacccgcg
     1801 ctatgacggc aataaaaaga cagaataaaa cgcacgggtg ttgggtcgtt tgttcataaa
     1861 cgcggggttc ggtcccaggg ctggcactct gtcgataccc caccgagacc ccattggggc
     1921 caatacgccc gcgtttcttc cttttcccca ccccaccccc caagttcggg tgaaggccca
     1981 gggctcgcag ccaacgtcgg ggcggcaggc cctgccatag cagatctgcg cagctggggc
     2041 tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     2101 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     2161 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg gggcatccct
     2221 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     2281 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     2341 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     2401 tattcttttg atttataagg gattttgggg atttcggcct attggttaaa aaatgagctg
     2461 atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta gggtgtggaa
     2521 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     2581 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     2641 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     2701 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     2761 ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     2821 tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa gagacaggat
     2881 gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg gccgcttggg
     2941 tggagaggct attcggctat gactgggcac aacagacaat cggctgctct gatgccgccg
     3001 tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac ctgtccggtg
     3061 ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg acgggcgttc
     3121 cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg ctattgggcg
     3181 aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa gtatccatca
     3241 tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca ttcgaccacc
     3301 aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt gtcgatcagg
     3361 atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc aggctcaagg
     3421 cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc ttgccgaata
     3481 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     3541 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     3601 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     3661 tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgcga aatgaccgac
     3721 caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg
     3781 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     3841 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     3901 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     3961 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     4021 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     4081 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     4141 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     4201 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     4261 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4321 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4381 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4441 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4501 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4561 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4621 gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4681 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4741 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4801 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4861 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4921 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4981 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     5041 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     5101 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     5161 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5221 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5281 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5341 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5401 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5461 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5521 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5581 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5641 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5701 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5761 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5821 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5881 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5941 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6001 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6061 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     6121 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     6181 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtcgact ctcagtacaa
     6241 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     6301 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
     6361 tgaagaatct gcttagg
//
