LOCUS       sequence-273235-        5908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of Nprl2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5908)
  AUTHORS   Shen K, Valenstein ML, Gu X, Sabatini DM
  TITLE     Arg-78 of Nprl2 catalyzes GATOR1-stimulated GTP hydrolysis by the 
            Rag GTPases.
  JOURNAL   J Biol Chem. 2019 Feb 22;294(8):2970-2975. doi: 
            10.1074/jbc.AC119.007382. Epub 2019 Jan 16.
  PUBMED    30651352
REFERENCE   2  (bases 1 to 5908)
  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: 
            "10.1074/jbc.AC119.007382"; journalName: "J Biol Chem"; date: 
            "2019-02-22- 22"; volume: "294"; issue: "8"; pages: "2970-2975"
COMMENT     Sequence Label: [pSK600] pRK5 HA-Nprl2(R78A)
FEATURES             Location/Qualifiers
     source          1..5908
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      963..972
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             975..1001
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      2208..2217
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2221..2355
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2258..2277)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2312..2331
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2424..2781
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2424..2615
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2447..2467)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2632..2767
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2801..2818)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2801..2817)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2810..2832)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3030..3485
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3117..3136)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3327..3348
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3502..3521)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3621..3643
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3681..3699)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3767..3871
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3872..3940,3941..4732)
                     /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     complement(4090..4109)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4903..5491
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5392..5411
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5645..5662
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5779..5800
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5815..5820,5821..5838,5839..5845)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5853..5869
                     /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)."
     primer_bind     5858..5880
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5877..5893
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5877..5893
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcccgaac cgacagtcgg tctcttcacc aaggccattc
      961 gcgccaccat gggctacccc tatgatgtgc ctgactacgc agatctcaat ggtggtggtg
     1021 gtgggtcgac gatgggcagc ggctgccgca tcgaatgcat attcttcagc gagttccacc
     1081 ccacgctggg acccaagatc acctatcagg tccctgaaga cttcatctcc cgagagctgt
     1141 ttgacacagt ccaagtgtac atcatcacca agccagagct gcagaacaag cttatcactg
     1201 tcacagctat ggaaaagaag ctgatcggct gtcctgtgtg catcgaacac aagaagtaca
     1261 gcgcgaatgc tctcctcttc aacctgggct tcgtgtgtga tgcccaggcc aagacctgcg
     1321 ccctcgagcc cattgttaaa aagctggctg gctatctgac cacactagag ctagagagca
     1381 gcttcgtgtc catggaggag agcaagcaga agttggtgcc catcatgacc atcttgctgg
     1441 aggagctaaa tgcctcaggc cggtgcactc tgcccattga tgagtccaac accatccact
     1501 tgaaggtgat tgagcagcgg ccagaccctc cggtggccca ggagtatgat gtacctgtct
     1561 ttaccaaaga caaggaggat ttcttcaact cacagtggga cctcactaca caacaaatcc
     1621 tgccctacat tgatgggttc cgccacatcc agaagatttc agcagaggca gatgtggagc
     1681 tcaacctggt gcgcattgct atccagaacc tgctgtacta cggcgttgtg acactggtgt
     1741 ccatcctcca gtactccaat gtatactgcc caacgcccaa ggtccaggac ctggtagatg
     1801 acaagtccct gcaagaggca tgtctatcct acgtgaccaa gcaagggcac aagagggcca
     1861 gtctccggga tgtgttccag ctatactgca gcctgagccc tggcactacc gtgcgagacc
     1921 tcattggccg ccacccccag cagctgcagc atgttgatga acggaagctg atccagttcg
     1981 ggcttatgaa gaacctcatc aggcgactac agaagtatcc tgtgcgggtg actcgggaag
     2041 agcagagcca ccctgcccgg ctttatacag gctgccacag ctatgacgag atctgctgca
     2101 agacaggcat gagctaccat gagctggatg agcggcttga aaatgacccc aacatcatca
     2161 tctgctggaa gtgagcggcc gctaagtaag taaggatccc cagcttggcc gccatggccc
     2221 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     2281 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     2341 tatcatgtct ggatcgggaa ttaattcggc gcagcaccat ggcctgaaat aacctctgaa
     2401 agaggaactt ggttaggtac cttctgaggc ggaaagaacc agctgtggaa tgtgtgtcag
     2461 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     2521 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2581 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     2641 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     2701 gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg aggctttttt
     2761 ggaggcctag gcttttgcaa aaagctgtta acagcttggc actggccgtc gttttacaac
     2821 gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca catccccctt
     2881 tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca
     2941 gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt
     3001 cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat taagcgcggc
     3061 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     3121 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     3181 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     3241 tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     3301 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     3361 ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3421 aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat taacgtttac
     3481 aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg
     3541 acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta
     3601 cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac cgtcatcacc
     3661 gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta atgtcatgat
     3721 aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg gaacccctat
     3781 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata
     3841 aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct
     3901 tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa cgctggtgaa
     3961 agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa
     4021 cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga tgagcacttt
     4081 taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg
     4141 tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca
     4201 tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa
     4261 cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa ccgctttttt
     4321 gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc
     4381 cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa
     4441 actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag actggatgga
     4501 ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct ggtttattgc
     4561 tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tggggccaga
     4621 tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga
     4681 acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga
     4741 ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat ttaaaaggat
     4801 ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg agttttcgtt
     4861 ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc ctttttttct
     4921 gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc
     4981 ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc
     5041 aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact ctgtagcacc
     5101 gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg gcgataagtc
     5161 gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc ggtcgggctg
     5221 aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata
     5281 cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta
     5341 tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag ggggaaacgc
     5401 ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg
     5461 atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt
     5521 cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt
     5581 ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga
     5641 gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac cgcctctccc
     5701 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     5761 cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc aggctttaca
     5821 ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat ttcacacagg
     5881 aaacagctat gacatgatta cgaattaa
//
