LOCUS       sequence-399423-        4888 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express N-terminal Tobacco Etch Virus (TEV) protease cleavable 
            Twin-Strep-tagged protein (ENLYFQGS-WSHPQFEK-(GGGS)2-GGSA-WSHPQFEK) 
            in mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4888)
  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 4888)
  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-C-TEV-Twin-Strep
FEATURES             Location/Qualifiers
     source          1..4888
                     /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             1772..1792
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1802..1825,1826..1861,1862..1885)
                     /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"
     CDS             1886..1906
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1910..1929)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    1975..2030
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2029..2048)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2391..2407)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2391..2407)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2404..2426)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2415..2431
                     /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(2439..2445,2446..2463,2464..2469))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2484..2505
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2564..2759
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2610..2745
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2765..2899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2802..2821)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2856..2875
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2967..2984)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3138..3726)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3218..3237)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3897..4688,4689..4757))
                     /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     4520..4539
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4758..4862)
                     /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 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 ggaaaacctc tattttcaag gcgccaccat
     1801 gtggtctcat ccccagttcg aaaaaggtgg tggaagtggt gggggctcag gaggatcagc
     1861 ctggtcacac cctcagttcg agaaggaaaa cctctatttt caaggcagat ctttttccct
     1921 ctgccaaaaa ttatggggac atcatgaagc cccttgagca tctgacttct ggctaataaa
     1981 ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca ctcggaagga
     2041 catatgggag ggcaaatcat ttaaaacatc agaatgagta tttggtttag agtttggcaa
     2101 catatgccca tatgctggct gccatgaaca aaggttggct ataaagaggt catcagtata
     2161 tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact tgaggttaga
     2221 ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa ttttccttac
     2281 atgttttact agccagattt ttcctcctct cctgactact cccagtcata gctgtccctc
     2341 ttctcttatg gagatccctc gacctgcagc ccaagcttgg cgtaatcatg gtcatagctg
     2401 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     2461 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     2521 ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga tccgcatctc aattagtcag
     2581 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     2641 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     2701 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa
     2761 agctaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     2821 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     2881 atcttatcat gtctggatcc gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     2941 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3001 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3061 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3121 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3181 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     3241 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     3301 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     3361 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     3421 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     3481 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     3541 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     3601 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     3661 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     3721 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     3781 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     3841 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     3901 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     3961 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4021 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4081 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4141 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4201 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     4261 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     4321 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     4381 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     4441 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     4501 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     4561 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     4621 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     4681 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     4741 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     4801 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     4861 cgcacatttc cccgaaaagt gccacctg
//
