LOCUS       sequence-445746-        7452 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Negative Control for downregulation of the expression of human DGK 
            alfa in human cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7452)
  AUTHORS   Avila-Flores A, Arranz-Nicolas J, Andrada E, Soutar D, Merida I
  TITLE     Predominant contribution of DGKzeta over DGKalpha in the control of 
            PKC/PDK-1-regulated functions in T cells.
  JOURNAL   Immunol Cell Biol. 2017 Jul;95(6):549-563. doi: 10.1038/icb.2017.7. 
            Epub 2017 Feb 6.
  PUBMED    28163304
REFERENCE   2  (bases 1 to 7452)
  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"; doi: 
            "10.1038/icb.2017.7"; journalName: "Immunol Cell Biol"; date: 
            "2017-07"; volume: "95"; issue: "6"; pages: "549-563"
COMMENT     Sequence Label: pSuper Retro GFP DGK alfa mouse
FEATURES             Location/Qualifiers
     source          1..7452
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     primer_bind     1421..1437
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1422..1438
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        complement(1511..1725)
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     complement(1571..1590)
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     promoter        1736..2239
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1771..1794)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2172..2191
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      2268..2277
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2274..2276,2277..2279,2280..2990)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2274..2276,2277..2279,2280..2990)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2319..2340)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2580..2599)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2927..2948
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3054..3848
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3108..3127)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3718..3737
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3912..3960
                     /note="synthetic polyadenylation signal"
     protein_bind    4521..4537
                     /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(4545..4551,4552..4569,4570..4575))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4590..4611
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4728..4745)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4899..5487)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4979..4998)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5658..6449,6450..6518))
                     /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     6281..6300
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6519..6623)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6691..6709
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6747..6769)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6869..6888
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     7082..7104
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7232..7251
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7451..7452,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatcgatctc tcgaggtcga cggtatcgat
     1441 aagcttttcc aaaaaacaca agaccacaga tgattctctt gaaatcatct gtggtcttgt
     1501 gtggggatct gtggtctcat acagaactta taagattccc aaatccaaag acatttcacg
     1561 tttatggtga tttcccagaa cacatagcga catgcaaata ttgcagggcg ccactcccct
     1621 gtccctcaca gccatcttcc tgccagggcg cacgcgcgct gggtgttccc gcctagtgac
     1681 actgggcccg cgattccttg gagcgggttg atgacgtcag cgttcgaatt ctaccgggta
     1741 ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact
     1801 tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg ccggtaggcg
     1861 ccaaccggct ccgttctttg gtggcccctt cgcgccacct tctactcctc ccctagtcag
     1921 gaagttcccc cccgccccgc agctcgcgtc gtgcaggacg tgacaaatgg aagtagcacg
     1981 tctcactagt ctcgtgcaga tggacagcac cgctgagcaa tggaagcggg taggcctttg
     2041 gggcagcggc caatagcagc tttgctcctt cgctttctgg gctcagaggc tgggaagggg
     2101 tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc
     2161 cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc
     2221 ctcatctccg ggcctttcga cctgcagccc aagctagctt accggtcgcc accatggtga
     2281 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
     2341 taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc
     2401 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     2461 ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg
     2521 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg
     2581 acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     2641 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     2701 agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca
     2761 aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact
     2821 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     2881 gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     2941 agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag tccggactca
     3001 gatcagcttg attcttctga cacaacagtc tcgaacttaa ggctagagcc accatgattg
     3061 aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta ttcggctatg
     3121 actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg tcagcgcagg
     3181 ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa ctgcaggacg
     3241 aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg
     3301 ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg caggatctcc
     3361 tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca atgcggcggc
     3421 tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat cgcatcgagc
     3481 gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac gaagagcatc
     3541 aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc gacggcgagg
     3601 atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa aatggccgct
     3661 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
     3721 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
     3781 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
     3841 tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca acctgccatc
     3901 acgatggccg caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg
     3961 aatcgatagc gataaggatc ggggatgcat cgataaaata aaagatttta tttagtctcc
     4021 agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg caagctagag aaccatcaga
     4081 tgtttccagg gtgccccaag gacctgaaat gaccctgtgc cttatttgaa ctaaccaatc
     4141 agttcgcttc tcgcttctgt tcgcgcgctt ctgctccccg agctcaataa aagagcccac
     4201 aacccctcac tcggcgcgcc agtcctccga tagactgcgt cgcccgggta cccgtgtatc
     4261 caataaaccc tcttgcagtt gcatccgact tgtggtctcg ctgttccttg ggagggtctc
     4321 ctctgagtga ttgactaccc gtcagcgggg gtctttcatg ggtaacagtt tcttgaagtt
     4381 ggagaacaac attctgaggg taggagtcga atattaagta atcctgactc aattagccac
     4441 tgttttgaat ccacatactc caatactcct gaaatagttc attatggaca gcgcagaaag
     4501 agctggggag aattgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga
     4561 agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg
     4621 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     4681 caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac
     4741 tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata
     4801 cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa
     4861 aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct
     4921 gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa
     4981 agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg
     5041 cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca
     5101 cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa
     5161 ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg
     5221 gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg
     5281 tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga
     5341 acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc
     5401 tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag
     5461 attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac
     5521 gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc
     5581 ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag
     5641 taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt
     5701 ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag
     5761 ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca
     5821 gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact
     5881 ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca
     5941 gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg
     6001 tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc
     6061 atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg
     6121 gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca
     6181 tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt
     6241 atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc
     6301 agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc
     6361 ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca
     6421 tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa
     6481 aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat
     6541 tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa
     6601 aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa
     6661 accattatta tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtctc
     6721 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     6781 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     6841 ggcgggtgtc ggggctggct taactatgcg gcatcagagc agattgtact gagagtgcac
     6901 catatgcggt gtgaaatacc gcacagatgc gtaaggagaa aataccgcat caggcgccat
     6961 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     7021 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
     7081 tcccagtcac gacgttgtaa aacgacggcg caaggaatgg tgcatgcaag gagatggcgc
     7141 ccaacagtcc cccggccacg gggcctgcca ccatacccac gccgaaacaa gcgctcatga
     7201 gcccgaagtg gcgagcccga tcttccccat cggtgatgtc ggcgatatag gcgccagcaa
     7261 ccgcacctgt ggcgccggtg atgccggcca cgatgcgtcc ggcgtagagg cgattagtcc
     7321 aatttgttaa agacaggata tcagtggtcc aggctctagt tttgactcaa caatatcacc
     7381 agctgaagcc tatagagtac gagccataga taaaataaaa gattttattt agtctccaga
     7441 aaaagggggg aa
//
