LOCUS       sequence-328068-        6695 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  FLCN expressing vector for mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6695)
  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 6695)
  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-FLCN
FEATURES             Location/Qualifiers
     source          1..6695
                     /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(3717..3736)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3782..3837
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3836..3855)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4198..4214)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4198..4214)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4211..4233)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4222..4238
                     /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(4246..4252,4253..4270,4271..4276))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4291..4312
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4371..4566
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4417..4552
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4572..4706
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4609..4628)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4663..4682
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4774..4791)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4945..5533)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5025..5044)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5704..6495,6496..6564))
                     /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     6327..6346
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6565..6669)
                     /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 sscsrggsgg ggsggggsgg ggsgrggggs
      541 ggggsggggs grggsggrgr ggkgsggcgg 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 grgggckcgg 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 aacgccatcg tggccctgtg ccacttctgt
     1921 gagctgcacg gcccacggac cctgttttgc acagaggtgc tgcacgcacc actgccacag
     1981 ggcgacggaa acgaggatag cccaggacag ggagagcagg cagaggagga ggagggcggc
     2041 atccagatga atagccggat gagagcccac tccccagcag agggagcctc tgtggagagc
     2101 tcctctcctg gcccaaagaa gagcgacatg tgcgagggat gtcggtccct ggccgcagga
     2161 caccctggct atatctctca cgataaggag acaagcatca agtacgtgtc tcaccagcac
     2221 cccagccacc ctcagctgtt ctccatcgtg cggcaggcct gcgtgagatc cctgtcttgc
     2281 gaggtgtgcc caggaagaga gggacctatc ttctttggcg acgagcagca cggcttcgtg
     2341 ttttctcaca ccttctttat caaggatagc ctggccaggg gcttccagcg gtggtattcc
     2401 atcatcacaa tcatgatgga ccggatctac ctgatcaact cttggccctt tctgctgggc
     2461 aaggtgagag gcatcatcga cgagctgcag ggcaaggccc tgaaggtgtt cgaggccgag
     2521 cagtttggct gtccacagag ggcccagagg atgaacaccg ccttcacacc ttttctgcac
     2581 cagaggaacg gcaatgccgc ccgctctctg accagcctga catccgacga taatctgtgg
     2641 gcctgcctgc acaccagctt tgcctggctg ctgaaggcct gtggctccag gctgacagag
     2701 aagctgctgg agggcgcccc taccgaggac acactggtgc agatggagaa gctggccgac
     2761 ctggaggagg agagcgagtc ctgggataat agcgaggcag aagaggagga gaaggcacca
     2821 gtgctgccag agtccaccga gggaagggag ctgacacagg gaccagccga gagctcctct
     2881 ctgtctggct gcggcagctg gcagcctaga aagctgccag tgttcaagtc tctgaggcac
     2941 atgagacagg tgctgggcgc ccccagcttt aggatgctgg cctggcacgt gctgatgggc
     3001 aaccaggtca tctggaagtc ccgcgacgtg gatctggtgc agtctgcctt cgaggtgctg
     3061 aggaccatgc tgccagtggg ctgcgtgcgc atcatcccct acagctccca gtatgaggag
     3121 gcctacaggt gtaatttcct gggcctgagc cctcacgtgc agatcccacc tcacgtgctg
     3181 tctagcgagt ttgccgtgat cgtggaggtg cacgccgccg cccggagcac cctgcaccca
     3241 gtgggctgtg aggacgatca gtctctgagc aagtatgagt ttgtggtgac atccggctct
     3301 cctgtggcag cagacagggt gggcccaacc atcctgaaca agatcgaggc cgccctgaca
     3361 aaccagaatc tgagcgtgga cgtggtggat cagtgcctgg tgtgcctgaa ggaggagtgg
     3421 atgaataagg tgaaggtgct gttcaagttt accaaggtgg actcccgccc taaggaggat
     3481 acacagaagc tgctgtccat cctgggcgcc tctgaggagg ataacgtgaa gctgctgaag
     3541 ttctggatga ccggcctgag caagacatac aagtcccacc tgatgtctac cgtgcggagc
     3601 ccaacagcca gcgagtccag aaattaactc gaggaattca ctcctcaggt gcaggctgcc
     3661 tatcagaagg tggtggctgg tgtggccaat gccctggctc acaaatacca ctgagatctt
     3721 tttccctctg ccaaaaatta tggggacatc atgaagcccc ttgagcatct gacttctggc
     3781 taataaagga aatttatttt cattgcaata gtgtgttgga attttttgtg tctctcactc
     3841 ggaaggacat atgggagggc aaatcattta aaacatcaga atgagtattt ggtttagagt
     3901 ttggcaacat atgcccatat gctggctgcc atgaacaaag gttggctata aagaggtcat
     3961 cagtatatga aacagccccc tgctgtccat tccttattcc atagaaaagc cttgacttga
     4021 ggttagattt tttttatatt ttgttttgtg ttattttttt ctttaacatc cctaaaattt
     4081 tccttacatg ttttactagc cagatttttc ctcctctcct gactactccc agtcatagct
     4141 gtccctcttc tcttatggag atccctcgac ctgcagccca agcttggcgt aatcatggtc
     4201 atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg
     4261 aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt
     4321 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagcggatcc gcatctcaat
     4381 tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt
     4441 tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc
     4501 gcctcggcct ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt
     4561 tgcaaaaagc taacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     4621 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     4681 tcaatgtatc ttatcatgtc tggatccgct gcattaatga atcggccaac gcgcggggag
     4741 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4801 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4861 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4921 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4981 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5041 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5101 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5161 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5221 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5281 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5341 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5401 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5461 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5521 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5581 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5641 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5701 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5761 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5821 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5881 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5941 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6001 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6061 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6121 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6181 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6241 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6301 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6361 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6421 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6481 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6541 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6601 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6661 ggttccgcgc acatttcccc gaaaagtgcc acctg
//
