LOCUS       sequence-196989-        5908 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of HA-Nprl2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5908)
  AUTHORS   Shen K, Choe A, Sabatini DM
  TITLE     Intersubunit Crosstalk in the Rag GTPase Heterodimer Enables mTORC1 
            to Respond Rapidly to Amino Acid Availability.
  JOURNAL   Mol Cell. 2017 Oct 17. pii: S1097-2765(17)30704-9. doi: 
            10.1016/j.molcel.2017.09.026.
  PUBMED    29056322
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"; journalName: "Mol Cell. 
            2017 Oct 17. pii: S1097-2765(17)30704-9. doi: 
            10.1016/j.molcel.2017.09.026."
COMMENT     Sequence Label: pRK5 HA-Nprl2
FEATURES             Location/Qualifiers
     source          1..5908
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..22
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(37..42,43..60,61..67)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    75..91
                     /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     80..102
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     99..115
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     99..115
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        144..523
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        524..727
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     678..698
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     724..748
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        958..976
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     958..975
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     989..1008
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1093..1102
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1105..1131
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      2338..2347
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2351..2485
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2388..2407)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2442..2461
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2554..2911
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2554..2745
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2577..2597)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2762..2897
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2931..2948)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2931..2947)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2940..2962)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3160..3615
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3247..3266)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3457..3478
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3632..3651)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3751..3773
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3811..3829)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3897..4001
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4002..4070,4071..4862)
                     /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(4220..4239)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5033..5621
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5522..5541
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5775..5792
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
       61 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgacatgat
      121 tacgaattaa ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat
      181 tacggggtca ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa
      241 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt
      301 tcccatagta acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta
      361 aactgcccac ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt
      421 caatgacggt aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc
      481 tacttggcag tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca
      541 gtacatcaat gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat
      601 tgacgtcaat gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa
      661 caactccgcc ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag
      721 cagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct
      781 ccatagaaga caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg
      841 gattccccgt gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt
      901 ggcttcgtta gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt
      961 taggtgacac tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag
     1021 gtccaactgc acctcggttc tatcgattga attcccgaac cgacagtcgg tctcttcacc
     1081 aaggccattc gcgccaccat gggctacccc tatgatgtgc ctgactacgc agatctcaat
     1141 ggtggtggtg gtgggtcgac gatgggcagc ggctgccgca tcgaatgcat attcttcagc
     1201 gagttccacc ccacgctggg acccaagatc acctatcagg tccctgaaga cttcatctcc
     1261 cgagagctgt ttgacacagt ccaagtgtac atcatcacca agccagagct gcagaacaag
     1321 cttatcactg tcacagctat ggaaaagaag ctgatcggct gtcctgtgtg catcgaacac
     1381 aagaagtaca gccgcaatgc tctcctcttc aacctgggct tcgtgtgtga tgcccaggcc
     1441 aagacctgcg ccctcgagcc cattgttaaa aagctggctg gctatctgac cacactagag
     1501 ctagagagca gcttcgtgtc catggaggag agcaagcaga agttggtgcc catcatgacc
     1561 atcttgctgg aggagctaaa tgcctcaggc cggtgcactc tgcccattga tgagtccaac
     1621 accatccact tgaaggtgat tgagcagcgg ccagaccctc cggtggccca ggagtatgat
     1681 gtacctgtct ttaccaaaga caaggaggat ttcttcaact cacagtggga cctcactaca
     1741 caacaaatcc tgccctacat tgatgggttc cgccacatcc agaagatttc agcagaggca
     1801 gatgtggagc tcaacctggt gcgcattgct atccagaacc tgctgtacta cggcgttgtg
     1861 acactggtgt ccatcctcca gtactccaat gtatactgcc caacgcccaa ggtccaggac
     1921 ctggtagatg acaagtccct gcaagaggca tgtctatcct acgtgaccaa gcaagggcac
     1981 aagagggcca gtctccggga tgtgttccag ctatactgca gcctgagccc tggcactacc
     2041 gtgcgagacc tcattggccg ccacccccag cagctgcagc atgttgatga acggaagctg
     2101 atccagttcg ggcttatgaa gaacctcatc aggcgactac agaagtatcc tgtgcgggtg
     2161 actcgggaag agcagagcca ccctgcccgg ctttatacag gctgccacag ctatgacgag
     2221 atctgctgca agacaggcat gagctaccat gagctggatg agcggcttga aaatgacccc
     2281 aacatcatca tctgctggaa gtgagcggcc gctaagtaag taaggatccc cagcttggcc
     2341 gccatggccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     2401 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     2461 caatgtatct tatcatgtct ggatcgggaa ttaattcggc gcagcaccat ggcctgaaat
     2521 aacctctgaa agaggaactt ggttaggtac cttctgaggc ggaaagaacc agctgtggaa
     2581 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     2641 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     2701 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     2761 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     2821 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     2881 aggctttttt ggaggcctag gcttttgcaa aaagctgtta acagcttggc actggccgtc
     2941 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     3001 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     3061 cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct tacgcatctg
     3121 tgcggtattt cacaccgcat acgtcaaagc aaccatagta cgcgccctgt agcggcgcat
     3181 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3241 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3301 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3361 ccaaaaaact tgatttgggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     3421 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     3481 caacactcaa ccctatctcg ggctattctt ttgatttata agggattttg ccgatttcgg
     3541 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     3601 taacgtttac aattttatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     3661 gccagccccg acacccgcca acacccgctg acgcgccctg acgggcttgt ctgctcccgg
     3721 catccgctta cagacaagct gtgaccgtct ccgggagctg catgtgtcag aggttttcac
     3781 cgtcatcacc gaaacgcgcg agacgaaagg gcctcgtgat acgcctattt ttataggtta
     3841 atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga aatgtgcgcg
     3901 gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc atgagacaat
     3961 aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt caacatttcc
     4021 gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct cacccagaaa
     4081 cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt tacatcgaac
     4141 tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt tttccaatga
     4201 tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtattgac gccgggcaag
     4261 agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac tcaccagtca
     4321 cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct gccataacca
     4381 tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg aaggagctaa
     4441 ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg gaaccggagc
     4501 tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgtagca atggcaacaa
     4561 cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa caattaatag
     4621 actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt ccggctggct
     4681 ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc attgcagcac
     4741 tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg agtcaggcaa
     4801 ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt aagcattggt
     4861 aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt catttttaat
     4921 ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc ccttaacgtg
     4981 agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct tcttgagatc
     5041 ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta ccagcggtgg
     5101 tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc ttcagcagag
     5161 cgcagatacc aaatactgtt cttctagtgt agccgtagtt aggccaccac ttcaagaact
     5221 ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct gctgccagtg
     5281 gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat aaggcgcagc
     5341 ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg acctacaccg
     5401 aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa gggagaaagg
     5461 cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg gagcttccag
     5521 ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga cttgagcgtc
     5581 gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc aacgcggcct
     5641 ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct gcgttatccc
     5701 ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct cgccgcagcc
     5761 gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca atacgcaaac
     5821 cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact
     5881 ggaaagcggg cagtgagcgc aacgcaat
//
