LOCUS       sequence-399646-        5975 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express C-terminal Tobacco Etch Virus (TEV) protease cleavable 
            Twin-Strep-tagged human Sphingomyelin synthase 2 (SMS2) in mammalian
            cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5975)
  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 5975)
  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-SMS2-TEV-Twin-Strep
FEATURES             Location/Qualifiers
     source          1..5975
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2877..2897
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(2907..2930,2931..2966,2967..2990)
                     /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(2997..3016)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3062..3117
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3116..3135)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3478..3494)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3478..3494)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3491..3513)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3502..3518
                     /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(3526..3532,3533..3550,3551..3556))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3571..3592
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3651..3846
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3697..3832
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3852..3986
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3889..3908)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3943..3962
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4054..4071)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4225..4813)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4305..4324)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4984..5775,5776..5844))
                     /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     5607..5626
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5845..5949)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggsgcgc gccrggsggg gsggggsggg gsgrggggsg
      541 gggsggggsg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gkgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg rgggctcggg ggaggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tgagctcccg
     1741 ggtaccgaat tcgatatcgc ggccgctcga gcaccatgga tatcatagag acagcaaaac
     1801 ttgaagaaca tttggaaaat caacccagtg atcctacgaa cacttatgca agacccgctg
     1861 aacctgttga agaagaaaac aaaaatggca atggtaaacc caagagctta tccagtgggc
     1921 tgcgaaaagg caccaaaaag tacccggact atatccaaat tgctatgccc actgaatcaa
     1981 ggaacaaatt tccactagag tggtggaaaa cgggcattgc cttcatatat gcagttttca
     2041 acctcgtctt gacaaccgtc atgatcacag ttgtacatga gagggtccct cccaaggagc
     2101 ttagccctcc actcccagac aagttttttg attacattga tagggtgaaa tgggcatttt
     2161 ctgtatcaga aataaatggg attatattag ttggattatg gatcacccag tggctgtttc
     2221 tgagatacaa gtcaatagtg ggacgcagat tctgttttat tattggaact ttatacctgt
     2281 atcgctgcat tacaatgtat gttactactc tacctgtgcc tggaatgcat ttccagtgtg
     2341 ctccaaagct caatggagac tctcaggcaa aagttcaacg gattctacga ttgatttctg
     2401 gtggtggatt gtccataact ggatcacata tcttatgtgg agacttcctc ttcagcggtc
     2461 acacggttac gctgacactg acttatttgt tcatcaaaga atattcgcct cgtcacttct
     2521 ggtggtatca tttaatctgc tggctgctga gtgctgccgg gatcatctgc attcttgtag
     2581 cacacgaaca ctacactatc gatgtgatca ttgcttatta tatcacaaca cgactgtttt
     2641 ggtggtacca ttcaatggcc aatgaaaaga acttgaaggt ctcttcacag actaatttct
     2701 tatctcgagc atggtggttc cccatctttt atttttttga gaaaaatgta caaggctcaa
     2761 ttccttgctg cttctcctgg ccgctgtctt ggcctcctgg ctgcttcaaa tcatcatgca
     2821 aaaagtattc acgggttcag aagattggtg aagacaatga gaaatcgacc ctcgaggaaa
     2881 acctctattt tcaaggcgcc accatgtggt ctcatcccca gttcgaaaaa ggtggtggaa
     2941 gtggtggggg ctcaggagga tcagcctggt cacaccctca gttcgagaag tgaagatctt
     3001 tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc
     3061 taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc
     3121 ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt
     3181 ttggcaacat atgcccatat gctggctgcc atgaacaaag gttggctata aagaggtcat
     3241 cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga
     3301 ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt
     3361 tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct
     3421 gtccctcttc tcttatggag atccctcgac ctgcagccca agcttggcgt aatcatggtc
     3481 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     3541 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     3601 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat
     3661 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     3721 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     3781 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     3841 tgcaaaaagc taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3901 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3961 tcaatgtatc ttatcatgtc tggatccgct gcattaatga atcggccaac gcgcggggag
     4021 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4081 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4141 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4201 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4261 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4321 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4381 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4441 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4501 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4561 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4621 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     4681 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4741 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4801 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4861 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4921 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4981 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5041 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5101 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5161 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5221 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5281 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5341 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5401 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5461 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5521 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5581 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5641 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5701 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5761 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5821 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5881 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5941 ggttccgcgc acatttcccc gaaaagtgcc acctg
//
