LOCUS       sequence-146866-        8203 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8203)
  AUTHORS   Tee AR, Fingar DC, Manning BD, Kwiatkowski DJ, Cantley LC, Blenis J
  TITLE     Tuberous sclerosis complex-1 and -2 gene products function together 
            to inhibit mammalian target of rapamycin (mTOR)-mediated downstream 
            signaling.
  JOURNAL   Proc Natl Acad Sci U S A. 2002 Oct 15. 99(21):13571-6.
  PUBMED    12271141
REFERENCE   2  (bases 1 to 8203)
  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"; journalName: "Proc Natl 
            Acad Sci U S A. 2002 Oct 15. 99(21):13571-6."
COMMENT     Sequence Label: pRK7-FLAG-TSC1
FEATURES             Location/Qualifiers
     source          1..8203
                     /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"
     CDS             933..956
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      4455..4464
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    4468..4602
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4505..4524)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4559..4578
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        4675..5032
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4675..4866
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4698..4718)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4883..5018
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4945..4964
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     complement(5052..5069)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5052..5068)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5061..5083)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5281..5736
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5368..5387)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5578..5599
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5753..5772)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5907..5929
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5982..6000)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        6068..6172
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6173..6241,6242..7033)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYM
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRDDAGQEQLGRRIHYSQNDLVEYS
                     PATEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6391..6410)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7204..7792
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7693..7712
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7946..7963
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        join(8116..8121,8122..8139,8140..8146)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8154..8170
                     /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     8159..8181
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8178..8194
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8178..8194
                     /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 taagcttctg caggtcgaca tggactataa ggacgatgat gacaaagccc
      961 aacaagcaaa tgtcggggag cttcttgcca tgctggactc ccccatgctg ggtgtgcggg
     1021 acgacgtgac agctgtcttt aaagagaacc tcaattctga ccgtggccct atgcttgtaa
     1081 acaccttggt ggattattac ctggaaacca gctctcagcc ggcattgcac atcctgacca
     1141 ccttgcaaga gccacatgac aagcacctct tggacaggat taacgaatat gtgggcaaag
     1201 ccgccactcg tttatccatc ctctcgttac tgggtcatgt cataagactg cagccatctt
     1261 ggaagcataa gctctctcaa gcacctcttt tgccttcttt actaaaatgt ctcaagatgg
     1321 acactgacgt cgttgtcctc acaacaggcg tcttggtgtt gataaccatg ctaccaatga
     1381 ttccacagtc tgggaaacag catcttcttg atttctttga catttttggc cgtctgtcat
     1441 catggtgcct gaagaaaccg ggccacgtgg cggaagtcta tcttgtccat ctccatgcca
     1501 gtgtgtacgc actctttcat cgcctttatg gaatgtaccc ttgcaacttc gtctcctttt
     1561 tgcgttctca ttacagtatg aaagaaaacc tggagacttt tgaagaagtg gtcaagccaa
     1621 tgatggagca tgtgcgaatt catccggaat tagtgactgg atccaaggac catgagctgg
     1681 accctcgaag gtggaagaga ttagaaactc atgatgttgt gatcgagtgt gccaaaatct
     1741 ctctggatcc cacagaagcc tcatatgaag atggctattc tgtgtctcac caaatctcag
     1801 cccgctttcc tcatcgttca gccgatgtca ccaccagccc ttatgctgac acacagaata
     1861 gctatgggtg tgctacttct accccttact ccacgtctcg gctgatgttg ttaaatatgc
     1921 cagggcagct acctcagact ctgagttccc catcgacacg gctgataact gaaccaccac
     1981 aagctactct ttggagccca tctatggttt gtggtatgac cactcctcca acttctcctg
     2041 gaaatgtccc acctgatctg tcacaccctt acagtaaagt ctttggtaca actgcaggtg
     2101 gaaaaggaac tcctctggga accccagcaa cctctcctcc tccagcccca ctctgtcatt
     2161 cggatgacta cgtgcacatt tcactccccc aggccacagt cacacccccc aggaaggaag
     2221 agagaatgga ttctgcaaga ccatgtctac acagacaaca ccatcttctg aatgacagag
     2281 gatcagaaga gccacctggc agcaaaggtt ctgtcactct aagtgatctt ccagggtttt
     2341 taggtgatct ggcctctgaa gaagatagta ttgaaaaaga taaagaagaa gctgcaatat
     2401 ctagagaact ttctgagatc accacagcag aggcagagcc tgtggttcct cgaggaggct
     2461 ttgactctcc cttttaccga gacagtctcc caggttctca gcggaagacc cactcggcag
     2521 cctccagttc tcagggcgcc agcgtgaacc ctgagccttt acactcctcc ctggacaagc
     2581 ttgggcctga cacaccaaag caagccttta ctcccataga cctgccctgc ggcagtgctg
     2641 atgaaagccc tgcgggagac agggaatgcc agacttcttt ggagaccagt atcttcactc
     2701 ccagtccttg taaaattcca cctccgacga gagtgggctt tggaagcggg cagcctcccc
     2761 cgtatgatca tctttttgag gtggcattgc caaagacagc ccatcatttt gtcatcagga
     2821 agactgagga gctgttaaag aaagcaaaag gaaacacaga ggaagatggt gtgccctcta
     2881 cctccccaat ggaagtgctg gacagactga tacagcaggg agcagacgcg cacagcaagg
     2941 agctgaacaa gttgccttta cccagcaagt ctgtcgactg gacccacttt ggaggctctc
     3001 ctccttcaga tgagatccgc accctccgag accagttgct tttactgcac aaccagttac
     3061 tctatgagcg ttttaagagg cagcagcatg ccctccggaa caggcggctc ctccgcaagg
     3121 tgatcaaagc agcagctctg gaggaacata atgctgccat gaaagatcag ttgaagttac
     3181 aagagaagga catccagatg tggaaggtta gtctgcagaa agaacaagct agatacaatc
     3241 agctccagga gcagcatgac actatggtaa ccaagctcca cagccagatc agacagctgc
     3301 agcatgaccg agaggaattc tacaaccaga gccaggaatt acagacgaag ctggaggact
     3361 gcaggaacat gattgcggag ctgcggatgg aactgaagaa ggccaacaac aaggtgtgtc
     3421 acactgagct gctgctcagt caggtttccc aaaagctctc aaacagtgag tcggtccagc
     3481 agcagatgga gttcttgaac aggcagctgt tggttcttgg ggaggtcaac gagctctatt
     3541 tggaacaact gcagaacaag cactcagata ccacaaagga agtagaaatg atgaaagccg
     3601 cctatcggaa agagctagaa aaaaacagaa gccatgttct ccagcagact cagaggcttg
     3661 atacctccca aaaacggatt ttggaactgg aatctcacct ggccaagaaa gaccaccttc
     3721 ttttggaaca gaagaaatat ctagaggatg tcaaactcca ggcaagagga cagctgcagg
     3781 ccgcagagag caggtatgag gctcagaaaa ggataaccca ggtgtttgaa ttggagatct
     3841 tagatttata tggcaggttg gagaaagatg gcctcctgaa aaaacttgaa gaagaaaaag
     3901 cagaagcagc tgaagcagca gaagaaaggc ttgactgttg taatgacggg tgctcagatt
     3961 ccatggtagg gcacaatgaa gaggcatctg gccacaacgg tgagaccaag acccccaggc
     4021 ccagcagcgc ccggggcagt agtggaagca gaggtggtgg aggcagcagc agcagcagca
     4081 gcgagctttc taccccagag aaacccccac accagagggc aggcccattc agcagtcggt
     4141 gggagacgac tatgggagaa gcgtctgcca gcatccccac cactgtgggc tcacttccca
     4201 gttcaaaaag cttcctgggt atgaaggctc gagagttatt tcgtaataag agcgagagcc
     4261 agtgtgatga ggacggcatg accagtagcc tttctgagag cctaaagaca gaactgggca
     4321 aagacttggg tgtggaagcc aagattcccc tgaacctaga tggccctcac ccgtctcccc
     4381 cgaccccgga cagtgttgga cagctacata tcatggacta caatgagact catcatgaac
     4441 acagctaatc gatggccgcc atggcccaac ttgtttattg cagcttataa tggttacaaa
     4501 taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt
     4561 ggtttgtcca aactcatcaa tgtatcttat catgtctgga tcgatcggga attaattcgg
     4621 cgcagcacca tggcctgaaa taacctctga aagaggaact tggttaggta ccttctgagg
     4681 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     4741 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     4801 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     4861 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     4921 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctgtga
     4981 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctgtt
     5041 aacagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     5101 caacttaatc gccttgcagc acatcccccc ttcgccacct ggcgtaatag cgaagaggcc
     5161 cgcaccgatc gcccttccca acagttgcgt agcctgaatg gcgaatggcg cctgatgcgg
     5221 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     5281 acgcgccctg tacgggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     5341 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     5401 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     5461 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     5521 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     5581 gactcttgtt ccaaactgga acaacactca accctatctc gggctattct tttgatttat
     5641 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     5701 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     5761 gctctgatgc cgcatagtta agccaactcc gctatcgcta cgtgactggg tcatggctgc
     5821 gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc
     5881 cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc
     5941 atcaccgaaa cgcgcgaggc agtattcttg aagacgaaag ggcctcgtga tacgcctatt
     6001 tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca cttttcgggg
     6061 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     6121 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat
     6181 tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc
     6241 tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg
     6301 ttacatggaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg
     6361 ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtgatga
     6421 cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta
     6481 ctcaccagcc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc
     6541 tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc
     6601 gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg
     6661 ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgccagcagc
     6721 aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca
     6781 acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct
     6841 tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat
     6901 cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg
     6961 gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgat
     7021 taagcattgg taactgtcag accaagttta ctcatatata ctttagattg atttaaaact
     7081 tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca tgaccaaaat
     7141 cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     7201 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     7261 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     7321 cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt taggccacca
     7381 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     7441 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     7501 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     7561 gacctacacc gaactgagat acctacagcg tgagcattga gaaagcgcca cgcttcccga
     7621 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     7681 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     7741 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     7801 caacgcggcc tttttacggt tcctggcctt ttggtggcct tttgctcaca tgttctttcc
     7861 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
     7921 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
     7981 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatccagct ggcacgacag
     8041 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt acctcactca
     8101 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     8161 cggataacaa tttcacacag gaaacagcta tgaccatgat tac
//
