LOCUS       sequence-174793-        6996 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 6996)
  AUTHORS   Wolfson RL, Chantranupong L, Wyant GA, Gu X, Orozco JM, Shen K, 
            Condon KJ, Petri S, Kedir J, Scaria SM, Abu-Remaileh M, Frankel WN, 
            Sabatini DM
  TITLE     KICSTOR recruits GATOR1 to the lysosome and is necessary for 
            nutrients to regulate mTORC1.
  JOURNAL   Nature. 2017 Feb 15. doi: 10.1038/nature21423.
  PUBMED    28199306
REFERENCE   2  (bases 1 to 6996)
  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: "Nature. 
            2017 Feb 15. doi: 10.1038/nature21423."
COMMENT     Sequence Label: plc242-GFP-Nprl2
FEATURES             Location/Qualifiers
     source          1..6996
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     LTR             1..470
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     misc_feature    535..734
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             935..1351
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1281..1303
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1319..1335
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     regulatory      1386..1395
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1392..1394,1395..1397,1398..2108)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1392..1394,1395..1397,1398..2108)
                     /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(1437..1458)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1698..1717)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2045..2066
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2154..2174
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             2187..2231
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(3574..3594)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3579..3908
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3759..3894
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3916..4314
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     LTR             4483..4952
                     /note="long terminal repeat from Moloney murine leukemia 
                     virus"
     primer_bind     complement(5082..5099)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5253..5841)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5333..5352)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6001..6792,6793..6861))
                     /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     6624..6643
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6862..6966)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctgaatatgg gccaaacagg atatctgtgg taagcagttc ctgccccggc tcagggccaa
       61 gaacagatgg aacagctgaa tatgggccaa acaggatatc tgtggtaagc agttcctgcc
      121 ccggctcagg gccaagaaca gatggtcccc agatgcggtc cagccctcag cagtttctag
      181 agaaccatca gatgtttcca gggtgcccca aggacctgaa atgaccctgt gccttatttg
      241 aactaaccaa tcagttcgct tctcgcttct gttcgcgcgc ttctgctccc cgagctcaat
      301 aaaagagccc acaacccctc actcggggcg ccagtcctcc gattgactga gtcgcccggg
      361 tacccgtgta tccaataaac cctcttgcag ttgcatccga cttgtggtct cgctgttcct
      421 tgggagggtc tcctctgagt gattgactac ccgtcagcgg gggtctttca tttgggggct
      481 cgtccgggat cgggagaccc ctgcccaggg accaccgacc caccaccggg aggcaagctg
      541 gccagcaact tatctgtgtc tgtccgattg tctagtgtct atgactgatt ttatgcgcct
      601 gcgtcggtac tagttagcta actagctctg tatctggcgg acccgtggtg gaactgacga
      661 gttctgaaca cccggccgca accctgggag acgtcccagg gactttgggg gccgtttttg
      721 tggcccgacc tgaggaaggg agtcgatgtg gaatccgacc ccgtcaggat atgtggttct
      781 ggtaggagac gagaacctaa aacagttccc gcctccgtct gaatttttgc tttcggtttg
      841 gaaccgaagc cgcgcgtctt gtctgctgca gcgctgcagc atcgttctgt gttgtctctg
      901 tctgactgtg tttctgtatt tgtctgaaaa ttagggccag actgttacca ctcccttaag
      961 tttgacctta ggtcactgga aagatgtcga gcggatcgct cacaaccagt cggtagatgt
     1021 caagaagaga cgttgggtta ccttctgctc tgcagaatgg ccaaccttta acgtcggatg
     1081 gccgcgagac ggcaccttta accgagacct catcacccag gttaagatca aggtcttttc
     1141 acctggcccg catggacacc cagaccaggt cccctacatc gtgacctggg aagccttggc
     1201 ttttgacccc cctccctggg tcaagccctt tgtacaccct aagcctccgc ctcctcttcc
     1261 tccatccgcc ccgtctctcc cccttgaacc tcctcgttcg accccgcctc gatcctccct
     1321 ttatccagcc ctcactcctt ctctaggcgc cggccggatc ccagtgtggt ggtacgctac
     1381 cggtcgccac catggtgagc aagggcgagg agctgttcac cggggtggtg cccatcctgg
     1441 tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag ggcgagggcg
     1501 atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag ctgcccgtgc
     1561 cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc cgctaccccg
     1621 accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc
     1681 gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg aagttcgagg
     1741 gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag gacggcaaca
     1801 tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc atggccgaca
     1861 agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag gacggcagcg
     1921 tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc gtgctgctgc
     1981 ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac gagaagcgcg
     2041 atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc atggacgagc
     2101 tgtacaagtc cggaggggat cggtggagct ccaccggtgg cggccgctct agagagaatc
     2161 tttattttca gggcgccgcc aaattcaaag aaaccgctgc tgctaaattc gaacgccagc
     2221 acatggacag cggaggtgga ggtactagtc tcgagatggg cagcggctgc cgcatcgaat
     2281 gcatattctt cagcgagttc caccccacgc tgggacccaa gatcacctat caggtccctg
     2341 aagacttcat ctcccgagag ctgtttgaca cagtccaagt gtacatcatc accaagccag
     2401 agctgcagaa caagcttatc actgtcacag ctatggaaaa gaagctgatc ggctgtcctg
     2461 tgtgcatcga acacaagaag tacagccgca atgctctcct cttcaacctg ggcttcgtgt
     2521 gtgatgccca ggccaagacc tgcgcccttg agcccattgt taaaaagctg gctggctatc
     2581 tgaccacact agagctagag agcagcttcg tgtccatgga ggagagcaag cagaagttgg
     2641 tgcccatcat gaccatcttg ctggaggagc taaatgcctc aggccggtgc actctgccca
     2701 ttgatgagtc caacaccatc cacttgaagg tgattgagca gcggccagac cctccggtgg
     2761 cccaggagta tgatgtacct gtctttacca aagacaagga ggatttcttc aactcacagt
     2821 gggacctcac tacacaacaa atcctgccct acattgatgg gttccgccac atccagaaga
     2881 tttcagcaga ggcagatgtg gagctcaacc tggtgcgcat tgctatccag aacctgctgt
     2941 actacggcgt tgtgacactg gtgtccatcc tccagtactc caatgtatac tgcccaacgc
     3001 ccaaggtcca ggacctggta gatgacaagt ccctgcaaga ggcatgtcta tcctacgtga
     3061 ccaagcaagg gcacaagagg gccagtctcc gggatgtgtt ccagctatac tgcagcctga
     3121 gccctggcac taccgtgcga gacctcattg gccgccaccc ccagcagctg cagcatgttg
     3181 atgaacggaa gctgatccag ttcgggctta tgaagaacct catcaggcga ctacagaagt
     3241 atcctgtgcg ggtgactcgg gaagagcaga gccaccctgc ccggctttat acaggctgcc
     3301 acagctatga cgagatctgc tgcaagacag gcatgagcta ccatgagctg gatgagcggc
     3361 ttgaaaatga ccccaacatc atcatctgct ggaagtgaga attctgcagt cgacggtacc
     3421 gcgggcccgg gatccaccgg atctagataa ctgatcataa tcagccatac cacatttgta
     3481 gaggttttac ttgctttaaa aaacctccca cacctccccc tgaacctgaa acataaaatg
     3541 aatgcaattc gccagcacag tggtcgaccc tgtggaatgt gtgtcagtta gggtgtggaa
     3601 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3661 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
     3721 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
     3781 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
     3841 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
     3901 tttgcaaaaa gcttcatggc caagcctttg tctcaagaag aatccaccct cattgaaaga
     3961 gcaacggcta caatcaacag catccccatc tctgaagact acagcgtcgc cagcgcagct
     4021 ctctctagcg acggccgcat cttcactggt gtcaatgtat atcattttac tgggggacct
     4081 tgtgcagaac tcgtggtgct gggcactgct gctgctgcgg cagctggcaa cctgacttgt
     4141 atcgtcgcga tcggaaatga gaacaggggc atcttgagcc cctgcggacg gtgccgacag
     4201 gtgcttctcg atctgcatcc tgggatcaaa gccatagtga aggacagtga tggacagccg
     4261 acggcagttg ggattcgtga attgctgccc tctggttatg tgtgggaggg ctaagatcga
     4321 taaaataaaa gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta
     4381 ggtttggcaa gctagcttaa gtaacgccat tttgcaaggc atggaaaaat acataactga
     4441 gaatagagaa gttcagatca aggtcaggaa cagatggaac agctgaatat gggccaaaca
     4501 ggatatctgt ggtaagcagt tcctgccccg gctcagggcc aagaacagat ggaacagctg
     4561 aatatgggcc aaacaggata tctgtggtaa gcagttcctg ccccggctca gggccaagaa
     4621 cagatggtcc ccagatgcgg tccagccctc agcagtttct agagaaccat cagatgtttc
     4681 cagggtgccc caaggacctg aaatgaccct gtgccttatt tgaactaacc aatcagttcg
     4741 cttctcgctt ctgttcgcgc gcttctgctc cccgagctca ataaaagagc ccacaacccc
     4801 tcactcgggg cgccagtcct ccgattgact gagtcgcccg ggtacccgtg tatccaataa
     4861 accctcttgc agttgcatcc gacttgtggt ctcgctgttc cttgggaggg tctcctctga
     4921 gtgattgact acccgtcagc gggggtcttt cacatgcagc atgtatcaaa attaatttgg
     4981 ttttttttct taagtattta cattaaatgg ccatagttgc attaatgaat cggccaacgc
     5041 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     5101 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     5161 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     5221 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     5281 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     5341 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     5401 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     5461 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     5521 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     5581 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     5641 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     5701 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     5761 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     5821 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     5881 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     5941 tagatccttt tgcggccgca aatcaatcta aagtatatat gagtaaactt ggtctgacag
     6001 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     6061 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     6121 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     6181 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     6241 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     6301 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     6361 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     6421 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     6481 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     6541 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6601 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6661 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6721 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6781 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6841 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6901 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6961 tccgcgcaca tttccccgaa aagtgccacc tgcagc
//
