LOCUS       sequence-305782-        6505 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  overexpression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6505)
  AUTHORS   Tsun ZY, Bar-Peled L, Chantranupong L, Zoncu R, Wang T, Kim C, 
            Spooner E, Sabatini DM
  TITLE     The folliculin tumor suppressor is a GAP for the RagC/D GTPases that
            signal amino acid levels to mTORC1.
  JOURNAL   Mol Cell. 2013 Nov 21;52(4):495-505. doi: 
            10.1016/j.molcel.2013.09.016. Epub 2013 Oct 3.
  PUBMED    24095279
REFERENCE   2  (bases 1 to 6505)
  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: 
            "10.1016/j.molcel.2013.09.016"; journalName: "Mol Cell"; date: 
            "2013-11-21- 21"; volume: "52"; issue: "4"; pages: "495-505"
COMMENT     Sequence Label: pRK5-HA-FLCN
FEATURES             Location/Qualifiers
     source          1..6505
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     581..605
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        815..833
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     815..832
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     846..865
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      950..959
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             962..988
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      2792..2801
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2805..2939
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2842..2861)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2896..2915
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3008..3365
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3008..3199
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3031..3051)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3216..3351
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3385..3402)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3385..3401)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3394..3416)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3614..4069
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3701..3720)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3911..3932
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4086..4105)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4205..4227
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4265..4283)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4351..4455
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4456..4524,4525..5316)
                     /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(4674..4693)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5487..6075
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5976..5995
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6229..6246
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6363..6384
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6399..6404,6405..6422,6423..6429)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6437..6453
                     /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     6442..6464
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6461..6477
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6461..6477
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctcgttta gtgaaccgtc
      601 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      661 ccagcctccg cggccgggaa cggtgcattg gaacgcggat tccccgtgcc aagagtgacg
      721 taagtaccgc ctatagagtc tataggccca cccccttggc ttcgttagaa cgcggctaca
      781 attaatacat aaccttatgt atcatacaca tacgatttag gtgacactat agaataacat
      841 ccactttgcc tttctctcca caggtgtcca ctcccaggtc caactgcacc tcggttctat
      901 cgattgaatt cccgaaccga cagtcggtct cttcaccaag gccattcgcg ccaccatggg
      961 ctacccctat gatgtgcctg actacgcaga tctcaatggt ggtggtggtg ggtcgacgat
     1021 gaatgccatc gtggctctct gccacttctg cgagctccac ggcccccgca ctctcttctg
     1081 cacggaggtg ctgcacgccc cacttcctca aggggatggg aatgaggaca gtcccggcca
     1141 gggtgagcag gcggaagaag aggaaggtgg cattcagatg aacagtcgga tgcgtgcgca
     1201 cagccccgca gagggggcca gcgtcgagtc cagcagcccg gggcccaaaa agtcggacat
     1261 gtgcgagggc tgccggtcac ttgctgcagg gcacccggga tatatcagcc atgataaaga
     1321 gacctccatt aaatacgtca gccaccagca ccccagccac ccccagctct tcagcattgt
     1381 ccgccaggcc tgtgtccgga gcctgagctg tgaggtctgc cctggccgtg aaggccccat
     1441 cttcttcgga gatgagcagc acggctttgt gttcagccac accttcttca tcaaggacag
     1501 cctggccagg ggcttccagc gctggtacag catcatcacc atcatgatgg accggatcta
     1561 cctcatcaac tcctggccct tcctgctggg gaaggtccgg ggaatcatcg atgagctcca
     1621 gggcaaggcg ctcaaggtgt ttgaggcaga gcagtttgga tgcccacagc gtgctcagag
     1681 gatgaacaca gccttcacgc cattcctaca ccagaggaac ggcaacgccg cccgctcgct
     1741 gacatcgctg acaagtgatg acaacctgtg ggcgtgcctg cacacctcct ttgcctggct
     1801 cctgaaggcg tgtggcagcc ggctgaccga gaagctcctg gaaggtgctc cgaccgagga
     1861 taccttggtc cagatggaga agctcgctga tttagaagag gaatcagaaa gctgggacaa
     1921 ctctgaggct gaagaggagg agaaagcccc tgtgttgcca gagagtacag aagggcggga
     1981 gctgacccag ggcccggcag agtcctcctc tctctcaggc tgtgggagct ggcagccccg
     2041 gaagctgcca gtcttcaagt ccctccggca catgaggcag gtcctgggtg ccccttcttt
     2101 ccgcatgctg gcctggcacg ttctcatggg gaaccaggtg atctggaaaa gcagagacgt
     2161 ggacctcgtc cagtcagctt ttgaagtact tcggaccatg cttcccgtgg gctgcgtccg
     2221 catcatccca tacagcagcc agtacgagga ggcctatcgg tgcaacttcc tggggctcag
     2281 cccgcacgtg cagatccccc cccacgtgct ctcctcagag tttgctgtca tcgtggaggt
     2341 ccacgcagcc gcacgttcca ccctccaccc tgtggggtgt gaggatgacc agtctctcag
     2401 caagtacgag tttgtggtga ccagtgggag ccctgtagct gcagaccgag tgggccccac
     2461 catcctgaat aagattgaag cggctctgac caaccagaac ctgtctgtgg atgtggtgga
     2521 ccagtgcctc gtctgcctca aggaggagtg gatgaacaaa gtgaaggtgc tttttaagtt
     2581 caccaaggtg gacagtcgcc ccaaagagga cacacagaag ctgctgagca tcctgggtgc
     2641 gtccgaggag gacaatgtca agctgctgaa gttctggatg actggcctga gcaagaccta
     2701 caagtcacac ctcatgtcca cggtccgcag ccccacagcc tcggagtctc ggaactgagc
     2761 ggccgctaag taagtaagga tccccagctt ggccgccatg gcccaacttg tttattgcag
     2821 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     2881 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcg
     2941 ggaattaatt cggcgcagca ccatggcctg aaataacctc tgaaagagga acttggttag
     3001 gtaccttctg aggcggaaag aaccagctgt ggaatgtgtg tcagttaggg tgtggaaagt
     3061 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     3121 ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg catctcaatt
     3181 agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     3241 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3301 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctaggctttt
     3361 gcaaaaagct gttaacagct tggcactggc cgtcgtttta caacgtcgtg actgggaaaa
     3421 ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa
     3481 tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg
     3541 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatacgtca
     3601 aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     3661 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     3721 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     3781 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt gggtgatggt
     3841 tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg
     3901 ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat ctcgggctat
     3961 tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt
     4021 taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt atggtgcact
     4081 ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc gccaacaccc
     4141 gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca agctgtgacc
     4201 gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg cgcgagacga
     4261 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     4321 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     4381 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     4441 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     4501 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     4561 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     4621 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     4681 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     4741 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     4801 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     4861 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     4921 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     4981 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     5041 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     5101 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     5161 ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     5221 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     5281 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     5341 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     5401 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     5461 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     5521 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     5581 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     5641 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     5701 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     5761 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     5821 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     5881 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     5941 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     6001 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     6061 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     6121 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     6181 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     6241 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     6301 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     6361 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
     6421 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgacatg
     6481 attacgaatt aattcgagct cgccc
//
