LOCUS       sequence-406370-        6130 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express C-terminal Tobacco Etch Virus (TEV) protease cleavable 
            Twin-Strep-tagged human Sphingomyelin synthase-relate protein (SMSr)
            in mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6130)
  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 6130)
  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-SMSr-TEV-Twin-Strep
FEATURES             Location/Qualifiers
     source          1..6130
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        385..662
                     /label=chicken beta-actin promoter
     intron          663..1679
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1603..1625
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1687..1706
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1774..1783
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3031..3051
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(3061..3084,3085..3120,3121..3144)
                     /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(3151..3170)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3216..3271
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3270..3289)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3632..3648)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3632..3648)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3645..3667)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3656..3672
                     /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(3680..3686,3687..3704,3705..3710))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3725..3746
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3805..4000
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3851..3986
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4006..4140
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4043..4062)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4097..4116
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4208..4225)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4379..4967)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4459..4478)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5138..5929,5930..5998))
                     /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     5761..5780
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5999..6103)
                     /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 ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc
      541 ggggcggggc gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt
      601 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
      661 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
      841 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
      901 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
     1021 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
     1081 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
     1141 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
     1201 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
     1261 cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg
     1321 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
     1381 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
     1441 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
     1501 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     1561 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     1621 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     1681 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttgagctccc
     1741 gggtaccgaa ttcgatatcg cggccgctcg agcgccacca tggcaggtcc taatcaactc
     1801 tgcattcgcc gctggactac caagcatgta gctgtgtggc tgaaggatga aggctttttt
     1861 gaatatgtgg acattttatg caataagcac cgacttgatg gaatcacatt gctaacattg
     1921 actgaatatg atctccggtc tcctcctctg gaaatcaaag tcttagggga cattaaaagg
     1981 ttaatgctct cagtccgaaa attgcagaaa atacatattg atgttttaga agagatgggc
     2041 tacaacagtg acagtcccat gggttccatg acccctttca tcagtgctct tcagagtaca
     2101 gactggctct gtaatgggga gctttcccat gactgtgacg gacccataac tgacttgaat
     2161 tctgatcagt accagtacat gaatggtaaa aacaaacatt ctgttcgaag attggaccca
     2221 gaatactgga agactatact gagttgtata tatgttttta tagtatttgg atttacatct
     2281 ttcattatgg ttatagtcca tgagcgagtg cctgacatgc agacctatcc accactccca
     2341 gatatattct tagacagcgt tcctagaatc ccatgggcct ttgccatgac ggaagtatgt
     2401 ggcatgattc tgtgctatat ttggctcctg gttcttcttc ttcacaagca caggtcaata
     2461 cttctgcgaa ggctctgtag tctgatggga actgtattct tgcttcgctg ctttaccatg
     2521 tttgtgacct ccctctccgt gccaggacaa cacctgcagt gtactggaaa gatatatggc
     2581 agtgtatggg agaaattaca tcgagccttt gccatttgga gtggctttgg tatgaccctg
     2641 actggcgttc acacatgtgg agattacatg tttagtggcc acacagtcgt cctaactatg
     2701 ctgaatttct ttgtcaccga atatacacca agaagctgga atttcttgca cactttatcc
     2761 tgggttctca acctctttgg aatcttcttc atcttggctg cccatgaaca ttattctatt
     2821 gatgtgttta ttgcttttta tataacaaca agactctttt tgtactacca tactctggcc
     2881 aataccagag catatcagca gagtaggaga gcaaggattt ggtttcccat gttctctttt
     2941 tttgaatgca atgttaatgg cacagtacct aatgaatatt gttggccatt ttcaaaacca
     3001 gcaataatga aaagactaat tggactcgag gaaaacctct attttcaagg cgccaccatg
     3061 tggtctcatc cccagttcga aaaaggtggt ggaagtggtg ggggctcagg aggatcagcc
     3121 tggtcacacc ctcagttcga gaagtgaaga tctttttccc tctgccaaaa attatgggga
     3181 catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc
     3241 aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca
     3301 tttaaaacat cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc
     3361 tgccatgaac aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt
     3421 ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt
     3481 tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac tagccagatt
     3541 tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct
     3601 cgacctgcag cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     3661 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     3721 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     3781 gaaacctgtc gtgccagcgg atccgcatct caattagtca gcaaccatag tcccgcccct
     3841 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     3901 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     3961 gtagtgagga ggcttttttg gaggcctagg cttttgcaaa aagctaactt gtttattgca
     4021 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     4081 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc
     4141 cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     4201 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     4261 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     4321 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     4381 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     4441 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4501 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4561 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4621 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4681 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4741 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4801 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4861 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4921 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4981 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5041 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     5101 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     5161 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     5221 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     5281 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     5341 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     5401 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     5461 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5521 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5581 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5641 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5701 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5761 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5821 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5881 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5941 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     6001 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     6061 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     6121 tgccacctgg
//
