LOCUS       sequence-327931-        6098 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  NPRL2 expressing vector for mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6098)
  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 6098)
  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-NPRL2
FEATURES             Location/Qualifiers
     source          1..6098
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          663..1679
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1603..1625
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1687..1706
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     regulatory      1753..1762
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1768..1791
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1828..1851
                     /codon_start=1
                     /product="peptide that binds Strep-Tactin(R), an engineered
                     form of streptavidin"
                     /label=Strep-Tag II
                     /translation="WSHPQFEK"
     CDS             1858..1881
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(3120..3139)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3185..3240
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3239..3258)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3601..3617)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3601..3617)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3614..3636)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3625..3641
                     /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(3649..3655,3656..3673,3674..3679))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3694..3715
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3774..3969
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3820..3955
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3975..4109
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4012..4031)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4066..4085
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4177..4194)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4348..4936)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4428..4447)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5107..5898,5899..5967))
                     /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     5730..5749
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5968..6072)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      421 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      481 cgatgggggc gggggggggg ggggggsgsg cscsrggsgg ggsggggsgg ggsgrggggs
      541 ggggsggggs grggsggrgr ggkgsggsgg cagccaatca gagcggcgcg ctccgaaagt
      601 ttccttttat ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg
      661 cgggagtcgc tgcgcgctgc cttcgccccg tgccccgctc cgccgccgcc tcgcgccgcc
      721 cgccccggct ctgactgacc gcgttactcc cacaggtgag cgggcgggac ggcccttctc
      781 ctccgggctg taattagcgc ttggtttaat gacggcttgt ttcttttctg tggctgcgtg
      841 aaagccttga ggggctccgg gagggccctt tgtgcggggg gagcggctcg gggggtgcgt
      901 gcgtgtgtgt gtgcgtgggg agcgccgcgt gcggctccgc gctgcccggc ggctgtgagc
      961 gctgcgggcg cggcgcgggg ctttgtgcgc tccgcagtgt gcgcgagggg agcgcggccg
     1021 ggggcggtgc cccgcggtgc ggggggggct gcgaggggaa caaaggctgc gtgcggggtg
     1081 tgtgcgtggg ggggtgagca gggggtgtgg gcgcgtcggt cgggctgcaa ccccccctgc
     1141 acccccctcc ccgagttgct gagcacggcc cggcttcggg tgcggggctc cgtacggggc
     1201 gtggcgcggg gctcgccgtg ccgggcgggg ggkggcggca ggkgggggkg ccgggcgggg
     1261 cggggccgcc ycgggcsggg grgggskcgg gggrggggcg cggcggcccc cggagcgccg
     1321 gcggctgtcg aggcgcggcg agccgcagcc attgcctttt atggtaatcg tgcgagaggg
     1381 cgcagggact tcctttgtcc caaatctgtg cggagccgaa atctgggagg cgccgccgca
     1441 ccccctctag cgggcgcggg gcgaagcggt gcggcgccgg caggaaggaa atgggcgggg
     1501 agggccttcg tgcgtcgccg cgccgccgtc cccttctccc tctccagcct cggggctgtc
     1561 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     1621 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     1681 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttcctcgatc
     1741 gacggtatcg atgccaccat ggctagctgg agccacccgc agttcgagaa aggtggaggt
     1801 gcccgaggtg gatcgggagg tggatcgtgg agccacccgc agttcgaaaa aggattcgat
     1861 tacaaggatg acgatgacaa gggtaccatg ggctccggct gcaggatcga gtgtatcttc
     1921 ttttctgagt tccaccctac cctgggccca aagatcacat accaggtgcc agaggacttc
     1981 atctcccgcg agctgtttga taccgtgcag gtgtatatca tcacaaagcc cgagctgcag
     2041 aacaagctga tcaccgtgac agctatggag aagaagctga tcggctgccc cgtgtgcatc
     2101 gagcacaaga agtactctcg gaacgccctg ctgttcaatc tgggcttcgt gtgcgacgcc
     2161 caggccaaga catgtgccct ggagccaatc gtgaagaagc tggccggcta tctgaccaca
     2221 ctggagctgg agagcagctt cgtgagcatg gaggagtcta agcagaagct ggtgcccatc
     2281 atgaccatcc tgctggagga gctgaacgcc agcggcaggt gcaccctgcc tatcgacgag
     2341 tccaatacaa tccacctgaa agtgatcgag cagcgccccg acccccctgt ggcacaggag
     2401 tacgatgtgc ccgtgttcac caaggacaag gaggatttct ttaactctca gtgggacctg
     2461 accacacagc agatcctgcc ctatatcgac ggctttcggc acatccagaa gatcagcgcc
     2521 gaggcagacg tggagctgaa cctggtgaga atcgccatcc agaatctgct gtactatggc
     2581 gtggtgacac tggtgagcat cctgcagtac tccaacgtgt actgcccaac ccccaaggtg
     2641 caggacctgg tggacgataa gtctctgcag gaggcctgtc tgagctacgt gaccaagcag
     2701 ggccacaagc gggcctccct gagagacgtg ttccagctgt attgttctct gagccctggc
     2761 accacagtga gggacctgat cggccgccac ccacagcagc tgcagcacgt ggatgagagg
     2821 aagctgatcc agtttggcct gatgaagaat ctgatccgga gactgcagaa gtacccagtg
     2881 agggtgaccc gcgaggagca gtctcaccca gccagactgt acacaggctg ccacagctat
     2941 gatgagatct gctgtaagac cggcatgtcc taccacgagc tggacgagag actggagaac
     3001 gatcctaata tcatcatctg ttggaagtaa ctcgaggaat tcactcctca ggtgcaggct
     3061 gcctatcaga aggtggtggc tggtgtggcc aatgccctgg ctcacaaata ccactgagat
     3121 ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca tctgacttct
     3181 ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca
     3241 ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta tttggtttag
     3301 agtttggcaa catatgccca tatgctggct gccatgaaca aaggttggct ataaagaggt
     3361 catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact
     3421 tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa
     3481 ttttccttac atgttttact agccagattt ttcctcctct cctgactact cccagtcata
     3541 gctgtccctc ttctcttatg gagatccctc gacctgcagc ccaagcttgg cgtaatcatg
     3601 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     3661 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     3721 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagcgga tccgcatctc
     3781 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
     3841 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
     3901 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
     3961 ttttgcaaaa agctaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4021 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4081 tcatcaatgt atcttatcat gtctggatcc gctgcattaa tgaatcggcc aacgcgcggg
     4141 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4201 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4261 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4321 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     4381 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     4441 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     4501 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     4561 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     4621 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     4681 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     4741 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     4801 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     4861 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     4921 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     4981 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5041 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5101 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5161 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5221 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5281 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     5341 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     5401 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     5461 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     5521 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     5581 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     5641 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     5701 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     5761 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     5821 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     5881 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     5941 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6001 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6061 aggggttccg cgcacatttc cccgaaaagt gccacctg
//
