LOCUS       sequence-273241-        6505 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  mammalian expression of FLCN.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6505)
  AUTHORS   Shen K, Rogala KB, Chou HT, Huang RK, Yu Z, Sabatini DM
  TITLE     Cryo-EM Structure of the Human FLCN-FNIP2-Rag-Ragulator Complex.
  JOURNAL   Cell. 2019 Nov 27;179(6):1319-1329.e8. doi: 
            10.1016/j.cell.2019.10.036. Epub 2019 Nov 6.
  PUBMED    31704029
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.cell.2019.10.036"; journalName: "Cell"; date: 
            "2019-11-27- 27"; volume: "179"; issue: "6"; pages: "1319-1329.e8"
COMMENT     Sequence Label: [pSK729] pRK5 HA-FLCN(R164A)
FEATURES             Location/Qualifiers
     source          1..6505
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        14..393
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        394..597
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     548..568
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     594..618
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        828..846
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     828..845
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     859..878
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      963..972
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             975..1001
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      2805..2814
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2818..2952
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2855..2874)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2909..2928
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        3021..3378
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3021..3212
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3044..3064)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3229..3364
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3398..3415)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3398..3414)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3407..3429)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3627..4082
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3714..3733)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3924..3945
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4099..4118)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4218..4240
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4278..4296)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4364..4468
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4469..4537,4538..5329)
                     /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(4687..4706)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5500..6088
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5989..6008
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6242..6259
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6376..6397
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6412..6417,6418..6435,6436..6442)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6450..6466
                     /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     6455..6477
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6474..6490
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6474..6490
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 ttcgagctcg cccgacattg attattgact agttattaat agtaatcaat tacggggtca
       61 ttagttcata gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct
      121 ggctgaccgc ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta
      181 acgccaatag ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac
      241 ttggcagtac atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt
      301 aaatggcccg cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag
      361 tacatctacg tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat
      421 gggcgtggat agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat
      481 gggagtttgt tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc
      541 ccattgacgc aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt
      601 ttagtgaacc gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga
      661 caccgggacc gatccagcct ccgcggccgg gaacggtgca ttggaacgcg gattccccgt
      721 gccaagagtg acgtaagtac cgcctataga gtctataggc ccaccccctt ggcttcgtta
      781 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac
      841 tatagaataa catccacttt gcctttctct ccacaggtgt ccactcccag gtccaactgc
      901 acctcggttc tatcgattga attcccgaac cgacagtcgg tctcttcacc aaggccattc
      961 gcgccaccat gggctacccc tatgatgtgc ctgactacgc agatctcaat ggtggtggtg
     1021 gtgggtcgac gatgaatgcc atcgtggctc tctgccactt ctgcgagctc cacggccccc
     1081 gcactctctt ctgcacggag gtgctgcacg ccccacttcc tcaaggggat gggaatgagg
     1141 acagtcccgg ccagggtgag caggcggaag aagaggaagg tggcattcag atgaacagtc
     1201 ggatgcgtgc gcacagcccc gcagaggggg ccagcgtcga gtccagcagc ccggggccca
     1261 aaaagtcgga catgtgcgag ggctgccggt cacttgctgc agggcacccg ggatatatca
     1321 gccatgataa agagacctcc attaaatacg tcagccacca gcaccccagc cacccccagc
     1381 tcttcagcat tgtccgccag gcctgtgtcc ggagcctgag ctgtgaggtc tgccctggcc
     1441 gtgaaggccc catcttcttc ggagatgagc agcacggctt tgtgttcagc cacaccttct
     1501 tcatcaagga cagcctggcc gctggcttcc agcgctggta cagcatcatc accatcatga
     1561 tggaccggat ctacctcatc aactcctggc ccttcctgct ggggaaggtc cggggaatca
     1621 tcgatgagct ccagggcaag gcgctcaagg tgtttgaggc agagcagttt ggatgcccac
     1681 agcgtgctca gaggatgaac acagccttca cgccattcct acaccagagg aacggcaacg
     1741 ccgcccgctc gctgacatcg ctgacaagtg atgacaacct gtgggcgtgc ctgcacacct
     1801 cctttgcctg gctcctgaag gcgtgtggca gccggctgac cgagaagctc ctggaaggtg
     1861 ctccgaccga ggataccttg gtccagatgg agaagctcgc tgatttagaa gaggaatcag
     1921 aaagctggga caactctgag gctgaagagg aggagaaagc ccctgtgttg ccagagagta
     1981 cagaagggcg ggagctgacc cagggcccgg cagagtcctc ctctctctca ggctgtggga
     2041 gctggcagcc ccggaagctg ccagtcttca agtccctccg gcacatgagg caggtcctgg
     2101 gtgccccttc tttccgcatg ctggcctggc acgttctcat ggggaaccag gtgatctgga
     2161 aaagcagaga cgtggacctc gtccagtcag cttttgaagt acttcggacc atgcttcccg
     2221 tgggctgcgt ccgcatcatc ccatacagca gccagtacga ggaggcctat cggtgcaact
     2281 tcctggggct cagcccgcac gtgcagatcc ccccccacgt gctctcctca gagtttgctg
     2341 tcatcgtgga ggtccacgca gccgcacgtt ccaccctcca ccctgtgggg tgtgaggatg
     2401 accagtctct cagcaagtac gagtttgtgg tgaccagtgg gagccctgta gctgcagacc
     2461 gagtgggccc caccatcctg aataagattg aagcggctct gaccaaccag aacctgtctg
     2521 tggatgtggt ggaccagtgc ctcgtctgcc tcaaggagga gtggatgaac aaagtgaagg
     2581 tgctttttaa gttcaccaag gtggacagtc gccccaaaga ggacacacag aagctgctga
     2641 gcatcctggg tgcgtccgag gaggacaatg tcaagctgct gaagttctgg atgactggcc
     2701 tgagcaagac ctacaagtca cacctcatgt ccacggtccg cagccccaca gcctcggagt
     2761 ctcggaactg agcggccgct aagtaagtaa ggatccccag cttggccgcc atggcccaac
     2821 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     2881 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     2941 catgtctgga tcgggaatta attcggcgca gcaccatggc ctgaaataac ctctgaaaga
     3001 ggaacttggt taggtacctt ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta
     3061 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3121 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3181 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3241 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3301 gaggccgagg ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3361 ggcctaggct tttgcaaaaa gctgttaaca gcttggcact ggccgtcgtt ttacaacgtc
     3421 gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat ccccctttcg
     3481 ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc
     3541 tgaatggcga atggcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3601 accgcatacg tcaaagcaac catagtacgc gccctgtagc ggcgcattaa gcgcggcggg
     3661 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     3721 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     3781 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3841 tttgggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3901 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3961 tatctcgggc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     4021 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgtttacaat
     4081 tttatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agccccgaca
     4141 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     4201 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     4261 acgcgcgaga cgaaagggcc tcgtgatacg cctattttta taggttaatg tcatgataat
     4321 aatggtttct tagacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     4381 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     4441 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     4501 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     4561 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     4621 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     4681 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     4741 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     4801 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     4861 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     4921 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     4981 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     5041 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     5101 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     5161 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     5221 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     5281 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     5341 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     5401 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     5461 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5521 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5581 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5641 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5701 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5761 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5821 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5881 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     5941 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6001 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     6061 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     6121 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     6181 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     6241 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     6301 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     6361 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     6421 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     6481 cagctatgac atgattacga attaa
//
