LOCUS       sequence-327932-        6666 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NPRL3 expressing vector for mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6666)
  AUTHORS   Lawrence RE, Fromm SA, Fu Y, Yokom AL, Kim DJ, Thelen AM, Young LN, 
            Lim CY, Samelson AJ, Hurley JH, Zoncu R
  TITLE     Structural mechanism of a Rag GTPase activation checkpoint by the 
            lysosomal folliculin complex.
  JOURNAL   Science. 2019 Nov 22;366(6468):971-977. doi: 
            10.1126/science.aax0364. Epub 2019 Oct 31.
  PUBMED    31672913
REFERENCE   2  (bases 1 to 6666)
  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: 
            "10.1126/science.aax0364"; journalName: "Science"; date: 
            "2019-11-22- 22"; volume: "366"; issue: "6468"; pages: "971-977"
COMMENT     Sequence Label: pCAG-TWIN-STREP-FLAG-NPRL3
FEATURES             Location/Qualifiers
     source          1..6666
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          664..1680
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1604..1626
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1688..1707
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1754..1763
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1769..1792
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1829..1852
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1859..1882
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3688..3707)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3753..3808
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3807..3826)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4169..4185)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4169..4185)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4182..4204)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4193..4209
                     /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(4217..4223,4224..4241,4242..4247))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4262..4283
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4342..4537
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4388..4523
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4543..4677
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4580..4599)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4634..4653
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4745..4762)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4916..5504)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4996..5015)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5675..6466,6467..6535))
                     /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     6298..6317
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6536..6640)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggggsgs gsscsrggsg gggsggggsg gggsgrgggg
      541 sggggsgggg sgrggsggrg rggkgsggsg gcagccaatc agagcggcgc gctccgaaag
      601 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
      661 gcgggagtcg ctgcgcgctg ccttcgcccc gtgccccgct ccgccgccgc ctcgcgccgc
      721 ccgccccggc tctgactgac cgcgttactc ccacaggtga gcgggcggga cggcccttct
      781 cctccgggct gtaattagcg cttggtttaa tgacggcttg tttcttttct gtggctgcgt
      841 gaaagccttg aggggctccg ggagggccct ttgtgcgggg ggagcggctc ggggggtgcg
      901 tgcgtgtgtg tgtgcgtggg gagcgccgcg tgcggctccg cgctgcccgg cggctgtgag
      961 cgctgcgggc gcggcgcggg gctttgtgcg ctccgcagtg tgcgcgaggg gagcgcggcc
     1021 gggggcggtg ccccgcggtg cggggggggc tgcgagggga acaaaggctg cgtgcggggt
     1081 gtgtgcgtgg gggggtgagc agggggtgtg ggcgcgtcgg tcgggctgca accccccctg
     1141 cacccccctc cccgagttgc tgagcacggc ccggcttcgg gtgcggggct ccgtacgggg
     1201 cgtggcgcgg ggctcgccgt gccgggcggg gggkggcggc aggkgggggk gccgggcggg
     1261 gcggggccgc cycgggcsgg ggrgggckcg ggggrggggc gcggcggccc ccggagcgcc
     1321 ggcggctgtc gaggcgcggc gagccgcagc cattgccttt tatggtaatc gtgcgagagg
     1381 gcgcagggac ttcctttgtc ccaaatctgt gcggagccga aatctgggag gcgccgccgc
     1441 accccctcta gcgggcgcgg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg
     1501 gagggccttc gtgcgtcgcc gcgccgccgt ccccttctcc ctctccagcc tcggggctgt
     1561 ccgcgggggg acggctgcct tcggggggga cggggcaggg cggggttcgg cttctggcgt
     1621 gtgaccggcg gctctagagc ctctgctaac catgttcatg ccttcttctt tttcctacag
     1681 ctcctgggca acgtgctggt tattgtgctg tctcatcatt ttggcaaaga attcctcgat
     1741 cgacggtatc gatgccacca tggctagctg gagccacccg cagttcgaga aaggtggagg
     1801 tgcccgaggt ggatcgggag gtggatcgtg gagccacccg cagttcgaaa aaggattcga
     1861 ttacaaggat gacgatgaca agggtaccat gagggacaac acaagcccca tctccgtgat
     1921 cctggtgagc tccggctccc gcggcaataa gctgctgttc aggtatcctt ttcagcgcag
     1981 ccaggagcac ccagcctctc agacaagcaa gcctcggtcc agatacgcag cctctaacac
     2041 cggcgaccac gcagatgagc aggacggcga ttccaggttc tctgatgtga tcctggccac
     2101 catcctggcc acaaagtccg agatgtgcgg ccagaagttc gagctgaaga tcgacaatgt
     2161 gcgctttgtg ggacacccca ccctgctgca gcacgcactg ggccagatca gcaagaccga
     2221 tccatcccca aagagggagg cacctacaat gatcctgttc aacgtggtgt ttgccctgag
     2281 agccaatgcc gacccatccg tgatcaactg cctgcacaat ctgtctcgga gaatcgccac
     2341 agtgctgcag cacgaggaga ggcgctgtca gtatctgacc agggaggcca agctgatcct
     2401 ggccctgcag gacgaggtga gcgcaatggc cgatggaaac gagggaccac agtccccttt
     2461 ccaccacatc ctgcctaagt gcaagctggc ccgggacctg aaggaggcct acgattccct
     2521 gtgcacctct ggcgtggtga gactgcacat caattcttgg ctggaggtga gcttttgcct
     2581 gccacacaag atccactatg cagcctctag cctgatccca cctgaggcaa tcgagcggag
     2641 cctgaaggcc atcagaccct accacgccct gctgctgctg agcgacgaga agtccctgct
     2701 gggcgagctg cctatcgact gttctcccgc cctggtgaga gtgatcaaga ccacaagcgc
     2761 cgtgaagaac ctgcagcagc tggcccagga cgcagatctg gccctgctgc aggtgtttca
     2821 gctggccgca cacctggtgt actggggcaa ggccatcatc atctatcctc tgtgcgagaa
     2881 caacgtgtac atgctgtctc ctaacgccag cgtgtgcctg tattccccac tggccgagca
     2941 gttctcccac cagtttccct ctcacgacct gcctagcgtg ctggccaagt tcagcctgcc
     3001 agtgtctctg agcgagttta gaaatccact ggccccagca gtgcaggaga cacagctgat
     3061 ccagatggtg gtgtggatgc tgcagcggag actgctgatc cagctgcaca cctacgtgtg
     3121 cctgatggcc tccccatctg aggaggagcc taggccacgc gaggacgatg tgcccttcac
     3181 cgccagggtg ggcggccgga gcctgtccac accaaacgcc ctgagctttg gctcccccac
     3241 ctcctctgac gatatgaccc tgacatctcc cagcatggac aatagctccg ccgagctgct
     3301 gccatctggc gatagccccc tgaaccagag gatgacagag aatctgctgg cctccctgtc
     3361 tgagcacgag cgggccgcaa tcctgagcgt gcctgcagcc cagaatccag aggatctgcg
     3421 gatgttcgcc agactgctgc actattttcg gggcagacac cacctggagg agatcatgta
     3481 caacgagaat accaggcgct cccagctgct gatgctgttc gacaagtttc ggtctgtgct
     3541 ggtggtgacc acacacgagg accccgtgat cgccgtgttc caggccctgc tgccttaact
     3601 cgaggaattc actcctcagg tgcaggctgc ctatcagaag gtggtggctg gtgtggccaa
     3661 tgccctggct cacaaatacc actgagatct ttttccctct gccaaaaatt atggggacat
     3721 catgaagccc cttgagcatc tgacttctgg ctaataaagg aaatttattt tcattgcaat
     3781 agtgtgttgg aattttttgt gtctctcact cggaaggaca tatgggaggg caaatcattt
     3841 aaaacatcag aatgagtatt tggtttagag tttggcaaca tatgcccata tgctggctgc
     3901 catgaacaaa ggttggctat aaagaggtca tcagtatatg aaacagcccc ctgctgtcca
     3961 ttccttattc catagaaaag ccttgacttg aggttagatt ttttttatat tttgttttgt
     4021 gttatttttt tctttaacat ccctaaaatt ttccttacat gttttactag ccagattttt
     4081 cctcctctcc tgactactcc cagtcatagc tgtccctctt ctcttatgga gatccctcga
     4141 cctgcagccc aagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4201 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     4261 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4321 acctgtcgtg ccagcggatc cgcatctcaa ttagtcagca accatagtcc cgcccctaac
     4381 tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact
     4441 aatttttttt atttatgcag aggccgaggc cgcctcggcc tctgagctat tccagaagta
     4501 gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctaacttgtt tattgcagct
     4561 tataatggtt acaaataaag caatagcatc acaaatttca caaataaagc atttttttca
     4621 ctgcattcta gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctggatccgc
     4681 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     4741 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4801 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4861 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4921 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4981 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     5041 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     5101 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     5161 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5221 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5281 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5341 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5401 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5461 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5521 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5581 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5641 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5701 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5761 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5821 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5881 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5941 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     6001 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     6061 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     6121 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6181 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6241 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6301 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6361 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6421 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6481 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6541 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6601 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6661 cacctg
//
