LOCUS       sequence-399624-        7681 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express N-terminal Tobacco Etch Virus (TEV) protease cleavable 
            Twin-Strep-tagged human diacylglycerol kinase zeta (DGK-zeta) in 
            mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7681)
  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 7681)
  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-Twin-Strep-TEV-DGK-zeta
FEATURES             Location/Qualifiers
     source          1..7681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(4..23)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    69..124
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(123..142)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(485..501)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(485..501)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(498..520)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    509..525
                     /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(533..539,540..557,558..563))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    578..599
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        658..853
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      704..839
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    859..993
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(896..915)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     950..969
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(1061..1078)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1232..1820)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1312..1331)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1991..2782,2783..2851))
                     /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     2614..2633
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2852..2956)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        2987..3366
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          3644..4660
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     4584..4606
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     4668..4687
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             4754..4774
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(4784..4807,4808..4843,4844..4867)
                     /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             4868..4888
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     regulatory      6749..6758
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
ORIGIN
        1 agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac
       61 ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct
      121 ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt
      181 ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag
      241 aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt
      301 gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct
      361 aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt
      421 catagctgtc cctcttctct tatggagatc cctcgacctg cagcccaagc ttggcgtaat
      481 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
      541 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
      601 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatccgca
      661 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
      721 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
      781 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
      841 aggcttttgc aaaaagctaa cttgtttatt gcagcttata atggttacaa ataaagcaat
      901 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
      961 aaactcatca atgtatctta tcatgtctgg atccgctgca ttaatgaatc ggccaacgcg
     1021 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     1081 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     1141 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     1201 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     1261 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     1321 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     1381 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     1441 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     1501 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     1561 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     1621 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     1681 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     1741 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     1801 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     1861 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     1921 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     1981 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     2041 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     2101 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     2161 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     2221 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     2281 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     2341 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     2401 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     2461 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     2521 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     2581 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     2641 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     2701 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     2761 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     2821 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     2881 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     2941 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgggtcgaca ttgattattg
     3001 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     3061 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     3121 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     3181 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     3241 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     3301 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     3361 accatggtcg aggtgagccc cacgttctgc ttcactctcc ccatctcccc cccctcccca
     3421 cccccaattt tgtatttatt tattttttaa ttattttgtg cagcgatggg ggcggggggg
     3481 gggggggggc gcgccrggsg gggsggggsg gggsgrgggg sggggsgggg sgrggcggag
     3541 aggtgcggcg gcagccaatc agagcggcgc gctccgaaag tttcctttta tggcgaggcg
     3601 gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg gcgggagtcg ctgcgcgctg
     3661 ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc ccgccccggc tctgactgac
     3721 cgcgttactc ccacaggtga gcgggcggga cggcccttct cctccgggct gtaattagcg
     3781 cttggtttaa tgacggcttg tttcttttct gtggctgcgt gaaagccttg aggggctccg
     3841 ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg tgcgtgtgtg tgtgcgtggg
     3901 gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag cgctgcgggc gcggcgcggg
     3961 gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc gggggcggtg ccccgcggtg
     4021 cggggggggc tgcgagggga acaaaggctg cgtgcggggt gtgtgcgtgg gggggtgagc
     4081 agggggtgtg ggcgcgtcgg tcgggctgca accccccctg cacccccctc cccgagttgc
     4141 tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg cgtggcgcgg ggctcgccgt
     4201 gccgggcggg gggtggcggc aggtgggggt gccgggcggg gcggggccgc ctcgggccgg
     4261 ggrgggctcg ggggaggggc gcggcggccc ccggagcgcc ggcggctgtc gaggcgcggc
     4321 gagccgcagc cattgccttt tatggtaatc gtgcgagagg gcgcagggac ttcctttgtc
     4381 ccaaatctgt gcggagccga aatctgggag gcgccgccgc accccctcta gcgggcgcgg
     4441 ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg gagggccttc gtgcgtcgcc
     4501 gcgccgccgt ccccttctcc ctctccagcc tcggggctgt ccgcgggggg acggctgcct
     4561 tcggggggga cggggcaggg cggggttcgg cttctggcgt gtgaccggcg gctctagagc
     4621 ctctgctaac catgttcatg ccttcttctt tttcctacag ctcctgggca acgtgctggt
     4681 tattgtgctg tctcatcatt ttggcaaaga attgagctcc cgggtaccga attcgatatc
     4741 gcggccgctc gaggaaaacc tctattttca aggcgccacc atgtggtctc atccccagtt
     4801 cgaaaaaggt ggtggaagtg gtgggggctc aggaggatca gcctggtcac accctcagtt
     4861 cgagaaggaa aacctctatt ttcaaggcag atctatggag ccgcgggacg gtagccccga
     4921 ggcccggagc agcgactccg agtcggcttc cgcctcgtcc agcggctccg agcgcgacgc
     4981 cggtcccgag ccggacaagg cgccgcggcg actcaacaag cggcgcttcc cggggctgcg
     5041 gctcttcggg cacaggaaag ccatcaccaa gtcgggcctc cagcacctgg ccccccctcc
     5101 gcccacccct ggggccccgt gcagcgagtc agagcggcag atccggagta cagtggactg
     5161 gagcgagtca gcgacatatg gggagcacat ctggttcgag accaacgtgt ccggggactt
     5221 ctgctacgtt ggggagcagt actgtgtagc caggatgctg aagtcagtgt ctcgaagaaa
     5281 gtgcgcagcc tgcaagattg tggtgcacac gccctgcatc gagcagctgg agaagataaa
     5341 tttccgctgt aagccgtcct tccgtgaatc aggctccagg aatgtccgcg agccaacctt
     5401 tgtacggcac cactgggtac acagacgacg ccaggacggc aagtgtcggc actgtgggaa
     5461 gggattccag cagaagttca ccttccacag caaggagatt gtggccatca gctgctcgtg
     5521 gtgcaagcag gcataccaca gcaaggtgtc ctgcttcatg ctgcagcaga tcgaggagcc
     5581 gtgctcgctg ggggtccacg cagccgtggt catcccgccc acctggatcc tccgcgcccg
     5641 gaggccccag aatactctga aagcaagcaa gaagaagaag agggcatcct tcaagaggaa
     5701 gtccagcaag aaagggcctg aggagggccg ctggagaccc ttcatcatca ggcccacccc
     5761 ctccccgctc atgaagcccc tgctggtgtt tgtgaacccc aagagtgggg gcaaccaggg
     5821 tgcaaagatc atccagtctt tcctctggta tctcaatccc cgacaagtct tcgacctgag
     5881 ccagggaggg cccaaggagg cgctggagat gtaccgcaaa gtgcacaacc tgcggatcct
     5941 ggcgtgcggg ggcgacggca cggtgggctg gatcctctcc accctggacc agctacgcct
     6001 gaagccgcca ccccctgttg ccatcctgcc cctgggtact ggcaacgact tggcccgaac
     6061 cctcaactgg ggtgggggct acacagatga gcctgtgtcc aagatcctct cccacgtgga
     6121 ggaggggaac gtggtacagc tggaccgctg ggacctccac gctgagccca accccgaggc
     6181 agggcctgag gaccgagatg aaggcgccac cgaccggttg cccctggatg tcttcaacaa
     6241 ctacttcagc ctgggctttg acgcccacgt caccctggag ttccacgagt ctcgagaggc
     6301 caacccagag aaattcaaca gccgctttcg gaataagatg ttctacgccg ggacagcttt
     6361 ctctgacttc ctgatgggca gctccaagga cctggccaag cacatccgag tggtgtgtga
     6421 tggaatggac ttgactccca agatccagga cctgaaaccc cagtgtgttg ttttcctgaa
     6481 catccccagg tactgtgcgg gcaccatgcc ctggggccac cctggggagc accacgactt
     6541 tgagccccag cggcatgacg acggctacct cgaggtcatt ggcttcacca tgacgtcgtt
     6601 ggccgcgctg caggtgggcg gacacggcga gcggctgacg cagtgtcgcg aggtggtgct
     6661 caccacatcc aaggccatcc cggtgcaggt ggatggcgag ccctgcaagc ttgcagcctc
     6721 acgcatccgc atcgccctgc gcaaccaggc caccatggtg cagaaggcca agcggcggag
     6781 cgccgccccc ctgcacagcg accagcagcc ggtgccagag cagttgcgca tccaggtgag
     6841 tcgcgtcagc atgcacgact atgaggccct gcactacgac aaggagcagc tcaaggaggc
     6901 ctctgtgccg ctgggcactg tggtggtccc aggagacagt gacctagagc tctgccgtgc
     6961 ccacattgag agactccagc aggagcccga tggtgctgga gccaagtccc cgacatgcca
     7021 gaaactgtcc cccaagtggt gcttcctgga cgccaccact gccagccgct tctacaggat
     7081 cgaccgagcc caggagcacc tcaactatgt gactgagatc gcacaggatg agatttatat
     7141 cctggaccct gagctgctgg gggcatcggc ccggcctgac ctcccaaccc ccacttcccc
     7201 tctccccacc tcaccctgct cacccacgcc ccggtcactg caaggggatg ctgcaccccc
     7261 tcaaggtgaa gagctgattg aggctgccaa gaggaacgac ttctgtaagc tccaggagct
     7321 gcaccgagct gggggcgacc tcatgcaccg agacgagcag agtcgcacgc tcctgcacca
     7381 cgcagtcagc actggcagca aggatgtggt ccgctacctg ctggaccacg cccccccaga
     7441 gatccttgat gcggtggagg aaaacgggga gacctgtttg caccaagcag cggccctggg
     7501 ccagcgcacc atctgccact acatcgtgga ggccggggcc tcgctcatga agacagacca
     7561 gcagggcgac actccccggc agcgggctga gaaggctcag gacaccgagc tggccgccta
     7621 cctggagaac cggcagcact accagatgat ccagcgggag gaccaggaga cggctgtgta
     7681 g
//
