LOCUS       sequence-393078-        6083 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 6083)
  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 6083)
  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..6083
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1670
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1594..1616
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     RBS             2254..2262
                     /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             3014..3034
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3107..3126)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3172..3227
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3226..3245)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3586..3602)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3586..3602)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3599..3621)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3610..3626
                     /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(3634..3640,3641..3658,3659..3664))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3679..3700
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3759..3954
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3805..3940
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3960..4094
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3997..4016)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4051..4070
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4162..4179)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4333..4921)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4413..4432)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5092..5883,5884..5952))
                     /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     5715..5734
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5953..6057)
                     /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 tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg ggagggcctt
     1501 cgtgcgtcgc cgcgccgccg tccccttctc catctccagc ctcggggctg ccgcaggggg
     1561 acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg
     1621 gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca
     1681 acgtgctggt tgttgtgctg tctcatcatt ttggcaaaga attgagctcc cgggtaccga
     1741 attcgatatc gcggccgctc gagccaccat gaaggaagtg gtttattggt cacccaagaa
     1801 ggtggcagac tggctgctgg agaatgctat gccagaatac tgtgagcctc tggagcattt
     1861 cacaggccag gacttgatca acctaaccca agaggatttc aaaaaacccc ccttgtgccg
     1921 agtctcctct gacaacgggc agcggctcct ggacatgata gaaaccctga aaatggagca
     1981 ccatttggaa gcacacaaga acggccatgc caatgggcac ctcaacattg gcgtagacat
     2041 ccccaccccc gacggcagct tcagcatcaa gattaaaccc aacgggatgc caaatgggta
     2101 taggaaagag atgataaaga tccccatgcc agaactggag cgctctcagt accccatgga
     2161 gtggggcaag acttttctgg cctttcttta tgcactttcc tgtttcgttc tcaccacagt
     2221 gatgatctcg gtcgtccacg aacgagtacc tcctaaggag gtgcagcctc cactaccgga
     2281 cacatttttt gaccatttta accgggtgca gtgggccttt tctatttgtg aaattaatgg
     2341 catgatcctt gtaggactct ggttaattca gtggctgctc ttaaaataca agtctattat
     2401 tagcagaaga tttttctgca tagttggcac gctgtacctg tatcggtgta ttacaatgta
     2461 tgtaactaca ctcccagtac ctggtatgca tttcaactgt tctccgaagc ttttcggaga
     2521 ctgggaagcc caactgcgaa gaataatgaa gctcattgct ggaggtggct tgtctatcac
     2581 tggctctcac aacatgtgtg gggactatct gtacagcggc cacacggtca tgctaacact
     2641 tacctactta tttatcaaag agtattcccc tcggcgactc tggtggtatc actggatttg
     2701 ctggcttctc agcgtagttg gaatcttctg tattctctta gcgcatgacc actacactgt
     2761 ggacgtggtg gtggcatatt acatcaccac gagactcttc tggtggtatc acactatggc
     2821 caatcagcaa gtgctaaagg aagcttccca gatgaacctc ctggccaggg tgtggtggta
     2881 caggccattt cagtactttg aaaagaatgt ccaaggaatt gtacctcgat cttaccattg
     2941 gcctttcccc tggccagtag tccacctcag taggcaagtt aaatacagcc ggctggtgaa
     3001 tgacacactc gaggaaaacc tctattttca aggcgccacc atgaaggatc atctcatcca
     3061 caatgtccac aaagaggagc acgctcatgc ccacaacaag tgaagatctt tttccctctg
     3121 ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga
     3181 aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat
     3241 atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat
     3301 atgccatatg ctggctgcca tgaacaaagg tggctataaa gaggtcatca gtatatgaaa
     3361 cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg ttagattttt
     3421 tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc cttacatgtt
     3481 ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt ccctcttctc
     3541 ttatgaagat ccctcgacct gcagcccaag cttggcgtaa tcatggtcat agctgtttcc
     3601 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     3661 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     3721 cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc
     3781 atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc cgcccattct
     3841 ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc ctcggcctct
     3901 gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg caaaaagcta
     3961 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     4021 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     4081 atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     4141 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     4201 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     4261 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     4321 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     4381 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     4441 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4501 cccttcggga agcgtggcgc tttctcaatg ctcacgctgt aggtatctca gttcggtgta
     4561 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4621 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4681 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4741 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     4801 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4861 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4921 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4981 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     5041 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     5101 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     5161 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     5221 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     5281 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     5341 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     5401 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     5461 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5521 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5581 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5641 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5701 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5761 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5821 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5881 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5941 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     6001 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     6061 atttccccga aaagtgccac ctg
//
