LOCUS       sequence-457432-        8344 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express human diacylglycerol kinase delta 2- SAM domain deletion 
            mutant (N-terminal TEV protease cleavable Twin-Strep-fusion protein)
            in mammalian cell.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8344)
  TITLE     PHOSPHO2 is PA phosphatase and PHOSPHO1 is PLC
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8344)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pCAGGS-Twin-strep-TEV-DGK-delta-2-DSAMD
FEATURES             Location/Qualifiers
     source          1..8344
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     87..109
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     171..190
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             257..277
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(287..310,311..346,347..370)
                     /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             371..391
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(3831..3850)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3896..3951
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3950..3969)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4312..4328)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4312..4328)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4325..4347)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4336..4352
                     /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(4360..4366,4367..4384,4385..4390))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4405..4426
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4485..4680
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4531..4666
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4686..4820
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4723..4742)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4777..4796
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4888..4905)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5059..5647)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5139..5158)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5818..6609,6610..6678))
                     /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     6441..6460
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6679..6783)
                     /gene="bla"
                     /label=AmpR promoter
     enhancer        6814..7193
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          join(7491..8344,1..163)
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
ORIGIN
        1 gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg
       61 ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag
      121 agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct
      181 ggttattgtg ctgtctcatc attttggcaa agaattgagc tcccgggtac cgaattcgat
      241 atcgcggccg ctcgaggaaa acctctattt tcaaggcgcc accatgtggt ctcatcccca
      301 gttcgaaaaa ggtggtggaa gtggtggggg ctcaggagga tcagcctggt cacaccctca
      361 gttcgagaag gaaaacctct attttcaagg cagatctgcg gcggcggcgg gcgcccctcc
      421 gccgggtccc ccgcaaccgc ctccgccgcc gccgcccgag gagtcgtccg acagcgagcc
      481 cgaggcggag cccggctccc cacagaagct catccgcaag gtgtccacgt cgggtcagat
      541 ccgacagaag accatcatca aagaggggat gctgaccaaa cagaacaatt cattccagcg
      601 atcaaaaagg agatacttta agcttcgagg gcgaacgctt tactatgcca aaacggcaaa
      661 gtcaatcata tttgatgagg tggatctgac agatgccagc gtagctgaat ccagtaccaa
      721 aaacgtcaac aacagtttta cggtcataac tccatgcagg aagctcatct tgtgtgctga
      781 taacagaaaa gaaatggaag attggattgc agcattaaag actgtgcaga acagggagca
      841 ctttgagccc acccagtaca gcatggacca cttctcaggg atgcacaatt ggtacgcctg
      901 ttcccacgcg aggccgacct actgcaatgt gtgccgtgag gctctgtctg gggtcacgtc
      961 gcacgggctg tcctgcgagg tgtgcaaatt taaggcccac aagcgctgtg ctgtgcgtgc
     1021 aaccaataac tgcaagtgga ccacactggc ctcgatcggg aaggacatca ttgaagatgc
     1081 agatgggatt gcaatgcccc accagtggtt ggaaggaaac ctacctgtga gcgccaagtg
     1141 cactgtgtgc gacaagacct gtggcagtgt gctgcgcctg caggactggc gctgcctctg
     1201 gtgcaaggcc atggttcaca catcgtgtaa agaatccttg ctgaccaagt gcccacttgg
     1261 cctgtgcaaa gtgtcagtca tcccacccac ggctctcaac agcatcgact ccgatgggtt
     1321 ctggaaggcc agctgtcctc cttcttgcac aagcccactg ttggtcttcg tcaattcaaa
     1381 aagtggggac aaccagggtg tgaagttcct cagaagattc aaacagctac taaaccccgc
     1441 ccaggtcttc gacctcatga acggaggccc acacctcggc ttacggttat tccagaagtt
     1501 tgacacattc cggattctgg tttgtggcgg ggatggaagt gttggctggg tcctctccga
     1561 aatcgacagc ctcaaccttc ataaacagtg tcagctggga gtgctgccgc tcggcacagg
     1621 gaacgacttg gcccgagtac tgggctgggg ctcagcctgc gatgacgaca cccagctccc
     1681 ccagatcttg gagaagttgg agagagccag caccaagatg ctggacaggt ggagcgtcat
     1741 ggcatacgag gccaagctcc cccggcaggc ctcctcctct accgtcaccg aagacttcag
     1801 cgaggattcc gaggtacagc agattctctt ctatgaagac tcggttgcag cccacctttc
     1861 taaaatcctc acctcggacc agcactcggt ggtcatctcc tcggccaaag tgctctgtga
     1921 gacggtgaag gacttcgtgg cacgggtggg gaaggcctat gagaagacga ccgagagctc
     1981 ggaggagtca gaggtcatgg ccaagaagtg ctctgtcctg aaagagaagc tggattccct
     2041 tctcaagacc ttggacgatg agtcccaggc ctcgtcctct ctgcccaacc cgccccccac
     2101 cattgccgag gaggctgaag atggagatgg gtcgggcagc atctgcggtt ccaccggaga
     2161 ccgcttggtg gcatcagctt gcccggcccg gccgcagata ttccggcctc gagaacagct
     2221 catgctgaga gccaacagcc tgaagaaagc aattcgtcag atcatagaac acacagaaaa
     2281 agctgtcgat gagcagaatg cccagaccca ggagcaggag ggcttcgtcc tgggcctctc
     2341 tgagtcagag gagaagatgg accacagagt gtgcccacca ctgtcccaca gcgagagctt
     2401 cggggtcccc aaggggagga gccagcgcaa agtgtcgaaa tctccgtgtg aaaagctgat
     2461 cagcaaaggg agtctgtccc taggcagttc tgcttccctt ccgccccagc cgggaagccg
     2521 ggacggcctg cctgcgctca acaccaagat cctgtaccca aatgtccggg ctggaatgtc
     2581 tggttcctta cccggtggct cagtcatcag tcgcctgtta attaatgctg atcccttcaa
     2641 ctctgaacca gaaaccctag agtattacac ggagaaatgt gtcatgaaca actattttgg
     2701 cattggcctg gatgcgaaga tatccctgga ctttaacaac aagcgcgatg agcacccaga
     2761 gaagtgcagg agccgaacca agaacatgat gtggtatgga gttcttggaa ccaaagagtt
     2821 gctgcacaga acctacaaga acctggagca aaaggtcttg ctggagtgtg acgggcgacc
     2881 catcccactc cccagtcttc agggaattgc tgtccttaac attcccagct atgccggagg
     2941 aaccaacttc tgggggggta ccaaggaaga tgatactttc gcagctccat cattcgatga
     3001 caagattctg gaggtggtcg ccgtgttcgg cagcatgcag atggccgtct ctcgagtcat
     3061 caggctacag catcatcgga tcgcccagtg tcgcacggtg aagatctcca tccttgggga
     3121 tgagggcgtg cctgtgcagg tggacggaga ggcctgggtc cagccgccag ggtacattcg
     3181 gattgtccac aagaaccggg cacagacact gaccagagac agggcatttg agagcaccct
     3241 gaagtcctgg gaagacaagc agaagtgcga gctgccccgc cctccatcct gttccctgca
     3301 cccggagatg ctgtccgagg aggaggccac ccagatggac cagtttgggc aggcagcagg
     3361 ggtcctcatt cacagtatcc gagaaatagc tcagtctcac cgggacatgg agcaggaact
     3421 ggcccacgcc gtcaatgcca gctccaagtc catggaccgt gtgtatggca agcccagaac
     3481 cacagagggg ctcaactgca gcttcgtcct ggaaatggtg aataacttca gagctctgcg
     3541 cagtgagacg gagctgctgc tgtctgggaa gatggccctg cagctggatc cgcctcagaa
     3601 ggagcagctg gggagtgctc ttgccgagat ggaccgacag ctcaggaggc tggcagacac
     3661 cccgtggctc tgccagtccg cagagcccgg cgacgaagag agtgtgatgc tggatcttgc
     3721 caagcgcagt cgcagtggta aattccgcct cgtgaccaag tttaaaaagg agaaaaacaa
     3781 caagaacaaa gaagctcaca gtagcctggg agccccggtt cacctctaga tctttttccc
     3841 tctgccaaaa attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa
     3901 aggaaattta ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg
     3961 acatatggga gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca
     4021 acatatgccc atatgctggc tgccatgaac aaaggttggc tataaagagg tcatcagtat
     4081 atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag
     4141 atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta
     4201 catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct
     4261 cttctcttat ggagatccct cgacctgcag cccaagcttg gcgtaatcat ggtcatagct
     4321 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     4381 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     4441 actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca
     4501 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     4561 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     4621 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     4681 aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     4741 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     4801 tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4861 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     4921 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4981 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5041 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5101 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5161 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5221 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5281 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5341 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     5401 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     5461 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     5521 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     5581 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     5641 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     5701 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     5761 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     5821 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     5881 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     5941 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     6001 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6061 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6121 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6181 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6241 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6301 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6361 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     6421 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     6481 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     6541 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     6601 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     6661 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     6721 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     6781 gcgcacattt ccccgaaaag tgccacctgg gtcgacattg attattgact agttattaat
     6841 agtaatcaat tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac
     6901 ttacggtaaa tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa
     6961 tgacgtatgt tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt
     7021 atttacggta aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc
     7081 ctattgacgt caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat
     7141 gggactttcc tacttggcag tacatctacg tattagtcat cgctattacc atggtcgagg
     7201 tgagccccac gttctgcttc actctcccca tctccccccc ctccccaccc ccaattttgt
     7261 atttatttat tttttaatta ttttgtgcag cgatgggggc gggggggggg gggggggggc
     7321 gcccgggggg gggggggggg gggggggggg gggggggggg gggggggggg gggggggcga
     7381 ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt ccttttatgg
     7441 cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg ggagtcgctg
     7501 cgcgctgcct tcgccccgtg ccccgctccg ccgccgcctc gcgccgcccg ccccggctct
     7561 gactgaccgc gttactccca caggtgagcg ggcgggacgg cccttctcct ccgggctgta
     7621 attagcgctt ggtttaatga cggcttgttt cttttctgtg gctgcgtgaa agccttgagg
     7681 ggctccggga gggccctttg tgcgggggga gcggctcggg gggtgcgtgc gtgtgtgtgt
     7741 gcgtggggag cgccgcgtgc ggctccgcgc tgcccggcgg ctgtgagcgc tgcgggcgcg
     7801 gcgcggggct ttgtgcgctc cgcagtgtgc gcgaggggag cgcggccggg ggcggtgccc
     7861 cgcggtgcgg ggggggctgc gaggggaaca aaggctgcgt gcggggtgtg tgcgtggggg
     7921 ggtgagcagg gggtgtgggc gcgtcggtcg ggctgcaacc ccccctgcac ccccctcccc
     7981 gagttgctga gcacggcccg gcttcgggtg cggggctccg tacggggcgt ggcgcggggc
     8041 tcgccgtgcc gggcgggggg tggcggcagg tgggggtgcc gggcggggcg gggccgcctc
     8101 gggccgggga gggctcgggg gaggggcgcg gcggcccccg gagcgccggc ggctgtcgag
     8161 gcgcggcgag ccgcagccat tgccttttat ggtaatcgtg cgagagggcg cagggacttc
     8221 ctttgtccca aatctgtgcg gagccgaaat ctgggaggcg ccgccgcacc ccctctagcg
     8281 ggcgcggggc gaagcggtgc ggcgccggca ggaaggaaat gggcggggag ggccttcgtg
     8341 cgtc
//
