LOCUS       sequence-399621-        6093 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express C-terminal Tobacco Etch Virus (TEV) protease cleavable 
            HAT-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 6093)
  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 6093)
  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-HAT
FEATURES             Location/Qualifiers
     source          1..6093
                     /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"
     RBS             2262..2270
                     /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             3022..3042
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3115..3134)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3180..3235
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3234..3253)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3596..3612)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3596..3612)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3609..3631)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3620..3636
                     /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(3644..3650,3651..3668,3669..3674))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3689..3710
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3769..3964
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3815..3950
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3970..4104
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4007..4026)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4061..4080
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4172..4189)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4343..4931)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4423..4442)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5102..5893,5894..5962))
                     /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     5725..5744
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5963..6067)
                     /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 gggggggcgc gccrggsggg gsggggsggg gsggggggsg
      541 gggsggggsg rggcggrgag gkgcggcggc 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 gtgggggtgc cgggcggggc
     1261 ggggccgcct cgggccgggg agggctcggg 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 gccaccatga aggaagtggt ttattggtca
     1801 cccaagaagg tggcagactg gctgctggag aatgctatgc cagaatactg tgagcctctg
     1861 gagcatttca caggccagga cttgatcaac ctaacccaag aggatttcaa aaaacccccc
     1921 ttgtgccgag tctcctctga caacgggcag cggctcctgg acatgataga aaccctgaaa
     1981 atggagcacc atttggaagc acacaagaac ggccatgcca atgggcacct caacattggc
     2041 gtagacatcc ccacccccga cggcagcttc agcatcaaga ttaaacccaa cgggatgcca
     2101 aatgggtata ggaaagagat gataaagatc cccatgccag aactggagcg ctctcagtac
     2161 cccatggagt ggggcaagac ttttctggcc tttctttatg cactttcctg tttcgttctc
     2221 accacagtga tgatctcggt cgtccacgaa cgagtacctc ctaaggaggt gcagcctcca
     2281 ctaccggaca cattttttga ccattttaac cgggtgcagt gggccttttc tatttgtgaa
     2341 attaatggca tgatccttgt aggactctgg ttaattcagt ggctgctctt aaaatacaag
     2401 tctattatta gcagaagatt tttctgcata gttggcacgc tgtacctgta tcggtgtatt
     2461 acaatgtatg taactacact cccagtacct ggtatgcatt tcaactgttc tccgaagctt
     2521 ttcggagact gggaagccca actgcgaaga ataatgaagc tcattgctgg aggtggcttg
     2581 tctatcactg gctctcacaa catgtgtggg gactatctgt acagcggcca cacggtcatg
     2641 ctaacactta cctacttatt tatcaaagag tattcccctc ggcgactctg gtggtatcac
     2701 tggatttgct ggcttctcag cgtagttgga atcttctgta ttctcttagc gcatgaccac
     2761 tacactgtgg ctgtggtggt ggcatattac atcaccacga gactcttctg gtggtatcac
     2821 actatggcca atcagcaagt gctaaaggaa gcttcccaga tgaacctcct ggccagggtg
     2881 tggtggtaca ggccatttca gtactttgaa aagaatgtcc aaggaattgt acctcgatct
     2941 taccattggc ctttcccctg gccagtagtc cacctcagta ggcaagttaa atacagccgg
     3001 ctggtgaatg acacactcga ggaaaacctc tattttcaag gcgccaccat gaaggatcat
     3061 ctcatccaca atgtccacaa agaggagcac gctcatgccc acaacaagtg aagatctttt
     3121 tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta
     3181 ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg
     3241 aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt
     3301 ggcaacatat gcccatatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca
     3361 gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg
     3421 ttagattttt tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc
     3481 cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt
     3541 ccctcttctc ttatggagat ccctcgacct gcagcccaag cttggcgtaa tcatggtcat
     3601 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     3661 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     3721 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta
     3781 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3841 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3901 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     3961 caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4021 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4081 aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag
     4141 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4201 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4261 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4321 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4381 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4441 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4501 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4561 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     4621 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     4681 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     4741 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     4801 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     4861 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     4921 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     4981 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5041 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5101 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5161 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     5221 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     5281 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     5341 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     5401 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     5461 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     5521 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     5581 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     5641 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     5701 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     5761 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     5821 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     5881 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     5941 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6001 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6061 ttccgcgcac atttccccga aaagtgccac ctg
//
