LOCUS       sequence-406371-        6119 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express C-terminal Tobacco Etch Virus (TEV) protease cleavable 
            Twin-Strep-tagged human Sphingomyelin synthase 1 (SMS1) in mammalian
            cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6119)
  AUTHORS   Furuta M, Murakami C, Numagami Y, Suzuki R, Sakane F
  TITLE     Diacylglycerol kinase zeta interacts with sphingomyelin synthase 1 
            and sphingomyelin synthase-related protein via different regions.
  JOURNAL   FEBS Open Bio. 2023 May 11. doi: 10.1002/2211-5463.13628.
  PUBMED    37166445
REFERENCE   2  (bases 1 to 6119)
  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: "FEBS Open 
            Bio. 2023 May 11. doi: 10.1002/2211-5463.13628."
COMMENT     Sequence Label: pCAGGS-SMS1-TEV-Twin-Strep
FEATURES             Location/Qualifiers
     source          1..6119
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..659
                     /label=chicken beta-actin promoter
     intron          660..1676
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1600..1622
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1684..1703
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     RBS             2260..2268
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     CDS             3020..3040
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(3050..3073,3074..3109,3110..3133)
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     primer_bind     complement(3140..3159)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3205..3260
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3259..3278)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3621..3637)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3621..3637)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3634..3656)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3645..3661
                     /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(3669..3675,3676..3693,3694..3699))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3714..3735
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3794..3989
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3840..3975
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3995..4129
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4032..4051)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4086..4105
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4197..4214)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4368..4956)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4448..4467)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5127..5918,5919..5987))
                     /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     5750..5769
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5988..6092)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg gggggcgcgc caggcggggc ggggcggggc gaggggcggg
      541 gcggggcgag gcggagaggt gcggcggcag ccaatcagag cggcgcgctc cgaaagtttc
      601 cttttatggc gaggcggcgg cggcggcggc cctataaaaa gcgaagcgcg cggcgggcgg
      661 gagtcgctgc gcgctgcctt cgccccgtgc cccgctccgc cgccgcctcg cgccgcccgc
      721 cccggctctg actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc
      781 cgggctgtaa ttagcgcttg gtttaatgac ggcttgtttc ttttctgtgg ctgcgtgaaa
      841 gccttgaggg gctccgggag ggccctttgt gcggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gctccgcgct gcccggcggc tgtgagcgct
      961 gcgggcgcgg cgcggggctt tgtgcgctcc gcagtgtgcg cgaggggagc gcggccgggg
     1021 gcggtgcccc gcggtgcggg gggggctgcg aggggaacaa aggctgcgtg cggggtgtgt
     1081 gcgtgggggg gtgagcaggg ggtgtgggcg cgtcggtcgg gctgcaaccc cccctgcacc
     1141 cccctccccg agttgctgag cacggcccgg cttcgggtgc ggggctccgt acggggcgtg
     1201 gcgcggggct cgccgtgccg ggcggggggt ggcggcaggt gggggtgccg ggcggggcgg
     1261 ggccgcctcg ggccggggag ggctcggggg aggggcgcgg cggcccccgg agcgccggcg
     1321 gctgtcgagg cgcggcgagc cgcagccatt gccttttatg gtaatcgtgc gagagggcgc
     1381 agggacttcc tttgtcccaa atctgtgcgg agccgaaatc tgggaggcgc cgccgcaccc
     1441 cctctagcgg gcgcggggcg aagcggtgcg gcgccggcag gaaggaaatg ggcggggagg
     1501 gccttcgtgc gtcgccgcgc cgccgtcccc ttctccctct ccagcctcgg ggctgtccgc
     1561 ggggggacgg ctgccttcgg gggggacggg gcagggcggg gttcggcttc tggcgtgtga
     1621 ccggcggctc tagagcctct gctaaccatg ttcatgcctt cttctttttc ctacagctcc
     1681 tgggcaacgt gctggttatt gtgctgtctc atcattttgg caaagaattg agctcccggg
     1741 taccgaattc gatatcgcgg ccgctcgagc caccatgaag gaagtggttt attggtcacc
     1801 caagaaggtg gcagactggc tgctggagaa tgctatgcca gaatactgtg agcctctgga
     1861 gcatttcaca ggccaggact tgatcaacct aacccaagag gatttcaaaa aacccccctt
     1921 gtgccgagtc tcctctgaca acgggcagcg gctcctggac atgatagaaa ccctgaaaat
     1981 ggagcaccat ttggaagcac acaagaacgg ccatgccaat gggcacctca acattggcgt
     2041 agacatcccc acccccgacg gcagcttcag catcaagatt aaacccaacg ggatgccaaa
     2101 tgggtatagg aaagagatga taaagatccc catgccagaa ctggagcgct ctcagtaccc
     2161 catggagtgg ggcaagactt ttctggcctt tctttatgca ctttcctgtt tcgttctcac
     2221 cacagtgatg atctcggtcg tccacgaacg agtacctcct aaggaggtgc agcctccact
     2281 accggacaca ttttttgacc attttaaccg ggtgcagtgg gccttttcta tttgtgaaat
     2341 taatggcatg atccttgtag gactctggtt aattcagtgg ctgctcttaa aatacaagtc
     2401 tattattagc agaagatttt tctgcatagt tggcacgctg tacctgtatc ggtgtattac
     2461 aatgtatgta actacactcc cagtacctgg tatgcatttc aactgttctc cgaagctttt
     2521 cggagactgg gaagcccaac tgcgaagaat aatgaagctc attgctggag gtggcttgtc
     2581 tatcactggc tctcacaaca tgtgtgggga ctatctgtac agcggccaca cggtcatgct
     2641 aacacttacc tacttattta tcaaagagta ttcccctcgg cgactctggt ggtatcactg
     2701 gatttgctgg cttctcagcg tagttggaat cttctgtatt ctcttagcgc atgaccacta
     2761 cactgtggac gtggtggtgg catattacat caccacgaga ctcttctggt ggtatcacac
     2821 tatggccaat cagcaagtgc taaaggaagc ttcccagatg aacctcctgg ccagggtgtg
     2881 gtggtacagg ccatttcagt actttgaaaa gaatgtccaa ggaattgtac ctcgatctta
     2941 ccattggcct ttcccctggc cagtagtcca cctcagtagg caagttaaat acagccggct
     3001 ggtgaatgac acactcgagg aaaacctcta ttttcaaggc gccaccatgt ggtctcatcc
     3061 ccagttcgaa aaaggtggtg gaagtggtgg gggctcagga ggatcagcct ggtcacaccc
     3121 tcagttcgag aagtgaagat ctttttccct ctgccaaaaa ttatggggac atcatgaagc
     3181 cccttgagca tctgacttct ggctaataaa ggaaatttat tttcattgca atagtgtgtt
     3241 ggaatttttt gtgtctctca ctcggaagga catatgggag ggcaaatcat ttaaaacatc
     3301 agaatgagta tttggtttag agtttggcaa catatgccca tatgctggct gccatgaaca
     3361 aaggttggct ataaagaggt catcagtata tgaaacagcc ccctgctgtc cattccttat
     3421 tccatagaaa agccttgact tgaggttaga ttttttttat attttgtttt gtgttatttt
     3481 tttctttaac atccctaaaa ttttccttac atgttttact agccagattt ttcctcctct
     3541 cctgactact cccagtcata gctgtccctc ttctcttatg gagatccctc gacctgcagc
     3601 ccaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     3661 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     3721 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     3781 tgccagcgga tccgcatctc aattagtcag caaccatagt cccgccccta actccgccca
     3841 tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga ctaatttttt
     3901 ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag tagtgaggag
     3961 gcttttttgg aggcctaggc ttttgcaaaa agctaacttg tttattgcag cttataatgg
     4021 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     4081 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcc gctgcattaa
     4141 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4201 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4261 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4321 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4381 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4441 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4501 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4561 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4621 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4681 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4741 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4801 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4861 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4921 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4981 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5041 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5101 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5161 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5221 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5281 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5341 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5401 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5461 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5521 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5581 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5641 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5701 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5761 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5821 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5881 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5941 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6001 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6061 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgg
//
