LOCUS       sequence-457431-        8536 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express human diacylglycerol kinase delta 2 protein (N-terminal 
            Tobacco Etch Virus (TEV) protease cleavable Twin-Strep-fusion 
            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 8536)
  TITLE     PHOSPHO2 is PA phosphatase and PHOSPHO1 is PLC
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 8536)
  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            "Unpublished"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
FEATURES             Location/Qualifiers
     source          1..8536
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          663..1679
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1603..1625
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1687..1706
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             1773..1793
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             join(1803..1826,1827..1862,1863..1886)
                     /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             1887..1907
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(5557..5576)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5622..5677
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5676..5695)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(6038..6054)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6038..6054)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6051..6073)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6062..6078
                     /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(6086..6092,6093..6110,6111..6116))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6131..6152
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6211..6406
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6257..6392
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6412..6546
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6449..6468)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6503..6522
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6614..6631)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6785..7373)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6865..6884)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7544..8335,8336..8404))
                     /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     8167..8186
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8405..8509)
                     /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 tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg ggggggcggg cgccgggggg gggggggggg gggggggggg
      541 gggggggggg gggggggggg gggggggggg gcgccacaag agggggcgcg ctccgaaagt
      601 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
      661 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
      841 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
      901 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
     1021 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
     1081 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
     1141 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
     1201 gtggcgcggg gctcgccgtg ccgggcgggg ggtggcggca ggtgggggtg ccgggcgggg
     1261 cggggccgcc tcgggccggg gagggctcgg gggaggggcg cggcggcccc cggagcgccg
     1321 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
     1381 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
     1441 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
     1501 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     1561 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     1621 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     1681 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttgagctccc
     1741 gggtaccgaa ttcgatatcg cggccgctcg aggaaaacct ctattttcaa ggcgccacca
     1801 tgtggtctca tccccagttc gaaaaaggtg gtggaagtgg tgggggctca ggaggatcag
     1861 cctggtcaca ccctcagttc gagaaggaaa acctctattt tcaaggcaga tctgcggcgg
     1921 cggcgggcgc ccctccgccg ggtcccccgc aaccgcctcc gccgccgccg cccgaggagt
     1981 cgtccgacag cgagcccgag gcggagcccg gctccccaca gaagctcatc cgcaaggtgt
     2041 ccacgtcggg tcagatccga cagaagacca tcatcaaaga ggggatgctg accaaacaga
     2101 acaattcatt ccagcgatca aaaaggagat actttaagct tcgagggcga acgctttact
     2161 atgccaaaac ggcaaagtca atcatatttg atgaggtgga tctgacagat gccagcgtag
     2221 ctgaatccag taccaaaaac gtcaacaaca gttttacggt cataactcca tgcaggaagc
     2281 tcatcttgtg tgctgataac agaaaagaaa tggaagattg gattgcagca ttaaagactg
     2341 tgcagaacag ggagcacttt gagcccaccc agtacagcat ggaccacttc tcagggatgc
     2401 acaattggta cgcctgttcc cacgcgaggc cgacctactg caatgtgtgc cgtgaggctc
     2461 tgtctggggt cacgtcgcac gggctgtcct gcgaggtgtg caaatttaag gcccacaagc
     2521 gctgtgctgt gcgtgcaacc aataactgca agtggaccac actggcctcg atcgggaagg
     2581 acatcattga agatgcagat gggattgcaa tgccccacca gtggttggaa ggaaacctac
     2641 ctgtgagcgc caagtgcact gtgtgcgaca agacctgtgg cagtgtgctg cgcctgcagg
     2701 actggcgctg cctctggtgc aaggccatgg ttcacacatc gtgtaaagaa tccttgctga
     2761 ccaagtgccc acttggcctg tgcaaagtgt cagtcatccc acccacggct ctcaacagca
     2821 tcgactccga tgggttctgg aaggccagct gtcctccttc ttgcacaagc ccactgttgg
     2881 tcttcgtcaa ttcaaaaagt ggggacaacc agggtgtgaa gttcctcaga agattcaaac
     2941 agctactaaa ccccgcccag gtcttcgacc tcatgaacgg aggcccacac ctcggcttac
     3001 ggttattcca gaagtttgac acattccgga ttctggtttg tggcggggat ggaagtgttg
     3061 gctgggtcct ctccgaaatc gacagcctca accttcataa acagtgtcag ctgggagtgc
     3121 tgccgctcgg cacagggaac gacttggccc gagtactggg ctggggctca gcctgcgatg
     3181 acgacaccca gctcccccag atcttggaga agttggagag agccagcacc aagatgctgg
     3241 acaggtggag cgtcatggca tacgaggcca agctcccccg gcaggcctcc tcctctaccg
     3301 tcaccgaaga cttcagcgag gattccgagg tacagcagat tctcttctat gaagactcgg
     3361 ttgcagccca cctttctaaa atcctcacct cggaccagca ctcggtggtc atctcctcgg
     3421 ccaaagtgct ctgtgagacg gtgaaggact tcgtggcacg ggtggggaag gcctatgaga
     3481 agacgaccga gagctcggag gagtcagagg tcatggccaa gaagtgctct gtcctgaaag
     3541 agaagctgga ttcccttctc aagaccttgg acgatgagtc ccaggcctcg tcctctctgc
     3601 ccaacccgcc ccccaccatt gccgaggagg ctgaagatgg agatgggtcg ggcagcatct
     3661 gcggttccac cggagaccgc ttggtggcat cagcttgccc ggcccggccg cagatattcc
     3721 ggcctcgaga acagctcatg ctgagagcca acagcctgaa gaaagcaatt cgtcagatca
     3781 tagaacacac agaaaaagct gtcgatgagc agaatgccca gacccaggag caggagggct
     3841 tcgtcctggg cctctctgag tcagaggaga agatggacca cagagtgtgc ccaccactgt
     3901 cccacagcga gagcttcggg gtccccaagg ggaggagcca gcgcaaagtg tcgaaatctc
     3961 cgtgtgaaaa gctgatcagc aaagggagtc tgtccctagg cagttctgct tcccttccgc
     4021 cccagccggg aagccgggac ggcctgcctg cgctcaacac caagatcctg tacccaaatg
     4081 tccgggctgg aatgtctggt tccttacccg gtggctcagt catcagtcgc ctgttaatta
     4141 atgctgatcc cttcaactct gaaccagaaa ccctagagta ttacacggag aaatgtgtca
     4201 tgaacaacta ttttggcatt ggcctggatg cgaagatatc cctggacttt aacaacaagc
     4261 gcgatgagca cccagagaag tgcaggagcc gaaccaagaa catgatgtgg tatggagttc
     4321 ttggaaccaa agagttgctg cacagaacct acaagaacct ggagcaaaag gtcttgctgg
     4381 agtgtgacgg gcgacccatc ccactcccca gtcttcaggg aattgctgtc cttaacattc
     4441 ccagctatgc cggaggaacc aacttctggg ggggtaccaa ggaagatgat actttcgcag
     4501 ctccatcatt cgatgacaag attctggagg tggtcgccgt gttcggcagc atgcagatgg
     4561 ccgtctctcg agtcatcagg ctacagcatc atcggatcgc ccagtgtcgc acggtgaaga
     4621 tctccatcct tggggatgag ggcgtgcctg tgcaggtgga cggagaggcc tgggtccagc
     4681 cgccagggta cattcggatt gtccacaaga accgggcaca gacactgacc agagacaggg
     4741 catttgagag caccctgaag tcctgggaag acaagcagaa gtgcgagctg ccccgccctc
     4801 catcctgttc cctgcacccg gagatgctgt ccgaggagga ggccacccag atggaccagt
     4861 ttgggcaggc agcaggggtc ctcattcaca gtatccgaga aatagctcag tctcaccggg
     4921 acatggagca ggaactggcc cacgccgtca atgccagctc caagtccatg gaccgtgtgt
     4981 atggcaagcc cagaaccaca gaggggctca actgcagctt cgtcctggaa atggtgaata
     5041 acttcagagc tctgcgcagt gagacggagc tgctgctgtc tgggaagatg gccctgcagc
     5101 tggatccgcc tcagaaggag cagctgggga gtgctcttgc cgagatggac cgacagctca
     5161 ggaggctggc agacaccccg tggctctgcc agtccgcaga gcccggcgac gaagagagtg
     5221 tgatgctgga tcttgccaag cgcagtcgca gtggtaaatt ccgcctcgtg accaagttta
     5281 aaaaggagaa aaacaacaag aacaaagaag ctcacagtag cctgggagcc ccggttcacc
     5341 tctgggggac agaggaggtt gctgcctggc tggagcacct cagtctctgt gagtataagg
     5401 acatcttcac acggcacgac atccggggct ctgagctcct gcacctggag cggagggacc
     5461 tcaaggacct gggcgtgacc aaggtgggcc acatgaagag gatcctgtgt ggcatcaagg
     5521 agctgagccg cagcgccccc gccgtcgagg cctagatctt tttccctctg ccaaaaatta
     5581 tggggacatc atgaagcccc ttgagcatct gacttctggc taataaagga aatttatttt
     5641 cattgcaata gtgtgttgga attttttgtg tctctcactc ggaaggacat atgggagggc
     5701 aaatcattta aaacatcaga atgagtattt ggtttagagt ttggcaacat atgcccatat
     5761 gctggctgcc atgaacaaag gttggctata aagaggtcat cagtatatga aacagccccc
     5821 tgctgtccat tccttattcc atagaaaagc cttgacttga ggttagattt tttttatatt
     5881 ttgttttgtg ttattttttt ctttaacatc cctaaaattt tccttacatg ttttactagc
     5941 cagatttttc ctcctctcct gactactccc agtcatagct gtccctcttc tcttatggag
     6001 atccctcgac ctgcagccca agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     6061 attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag tgtaaagcct
     6121 ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc
     6181 agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat tagtcagcaa ccatagtccc
     6241 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     6301 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     6361 ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc taacttgttt
     6421 attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca
     6481 tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc
     6541 tggatccgct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     6601 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     6661 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     6721 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     6781 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     6841 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     6901 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     6961 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     7021 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     7081 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     7141 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     7201 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     7261 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     7321 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     7381 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     7441 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     7501 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     7561 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     7621 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     7681 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     7741 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     7801 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     7861 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     7921 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     7981 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     8041 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     8101 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     8161 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     8221 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     8281 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     8341 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     8401 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     8461 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     8521 gaaaagtgcc acctgg
//
