LOCUS       sequence-242547-        6042 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 6042)
  AUTHORS   Sancak Y, Thoreen CC, Peterson TR, Lindquist RA, Kang SA, Spooner E,
            Carr SA, Sabatini DM
  TITLE     PRAS40 is an insulin-regulated inhibitor of the mTORC1 protein 
            kinase.
  JOURNAL   Mol Cell. 2007 Mar 23. 25(6):903-15.
  PUBMED    17386266
REFERENCE   2  (bases 1 to 6042)
  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: "Mol Cell. 
            2007 Mar 23. 25(6):903-15."
COMMENT     Sequence Label: pRK5 HA GST Rheb1
FEATURES             Location/Qualifiers
     source          1..6042
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    128..149
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(164..169,170..187,188..194)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    202..218
                     /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     207..229
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     226..242
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     226..242
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        271..650
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        651..854
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     805..825
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     851..875
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        1085..1103
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1085..1102
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     1116..1135
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     regulatory      1220..1229
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1232..1258
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             1289..1942
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     1900..1922
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     regulatory      2599..2608
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    2612..2746
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2649..2668)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2703..2722
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        2815..3172
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2815..3006
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2838..2858)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3023..3158
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3192..3209)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3192..3208)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3201..3223)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3421..3876
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3508..3527)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3718..3739
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3893..3912)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4012..4034
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4072..4090)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4158..4262
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4263..4331,4332..5123)
                     /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(4481..4500)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5294..5882
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5783..5802
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     join(6036..6042,1..11)
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc gcgcgttggc
       61 cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc agtgagcgca
      121 acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac tttatgcttc
      181 cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga aacagctatg
      241 acatgattac gaattaattc gagctcgccc gacattgatt attgactagt tattaatagt
      301 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
      361 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
      421 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      481 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      541 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      601 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      661 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      721 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      781 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      841 atataagcag agctcgttta gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt
      901 ttgacctcca tagaagacac cgggaccgat ccagcctccg cggccgggaa cggtgcattg
      961 gaacgcggat tccccgtgcc aagagtgacg taagtaccgc ctatagagtc tataggccca
     1021 cccccttggc ttcgttagaa cgcggctaca attaatacat aaccttatgt atcatacaca
     1081 tacgatttag gtgacactat agaataacat ccactttgcc tttctctcca caggtgtcca
     1141 ctcccaggtc caactgcacc tcggttctat cgattgaatt cccgaaccga cagtcggtct
     1201 cttcaccaag gccattcgcg ccaccatggg ctacccctat gatgtgcctg actacgcaga
     1261 tctcaatggt ggtggtggtg ggtcgagaat gtcccctata ctaggttatt ggaaaattaa
     1321 gggccttgtg caacccactc gacttctttt ggaatatctt gaagaaaaat atgaagagca
     1381 tttgtatgag cgcgatgaag gtgataaatg gcgaaacaaa aagtttgaat tgggtttgga
     1441 gtttcccaat cttccttatt atattgatgg tgatgttaaa ttaacacagt ctatggccat
     1501 catacgttat atagctgaca agcacaacat gttgggtggt tgtccaaaag agcgtgcaga
     1561 gatttcaatg cttgaaggag cggttttgga tattagatac ggtgtttcga gaattgcata
     1621 tagtaaagac tttgaaactc tcaaagttga ttttcttagc aagctacctg aaatgctgaa
     1681 aatgttcgaa gatcgtttat gtcataaaac atatttaaat ggtgatcatg taacccatcc
     1741 tgacttcatg ttgtatgacg ctcttgatgt tgttttatac atggacccaa tgtgcctgga
     1801 tgcgttccca aaattagttt gttttaaaaa acgtattgaa gctatcccac aaattgataa
     1861 gtacttgaaa tccagcaagt atatagcatg gcctttgcag ggctggcaag ccacgtttgg
     1921 tggtggcgac catcctccaa aaggtggagg tggtgggtcg aggggaggtt ctggtggatc
     1981 aggaggttca ggtggatctg ggtcgacgat gccgcagtcc aagtcccgga agatcgcgat
     2041 cctgggctac cggtctgtgg ggaaatcctc attgacgatt caatttgttg aaggccaatt
     2101 tgtggactcc tacgatccaa ccatagaaaa cacttttaca aagttgatca cagtaaatgg
     2161 acaagaatat catcttcaac ttgtagacac agccgggcaa gatgaatatt ctatctttcc
     2221 tcagacatac tccatagata ttaatggcta tattcttgtg tattctgtta catcaatcaa
     2281 aagttttgaa gtgattaaag ttatccatgg caaattgttg gatatggtgg ggaaagtaca
     2341 aatacctatt atgttggttg ggaataagaa agacctgcat atggaaaggg tgatcagtta
     2401 tgaagaaggg aaagctttgg cagaatcttg gaatgcagct tttttggaat cttctgctaa
     2461 agaaaatcag actgctgtgg atgtttttcg aaggataatt ttggaggcag aaaaaatgga
     2521 cggggcagct tcacaaggga agtcttcgtg ctcggtgatg tgaatgcggc cgctaagtaa
     2581 gtaaggatcc ccagcttggc cgccatggcc caacttgttt attgcagctt ataatggtta
     2641 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     2701 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcggga attaattcgg
     2761 cgcagcacca tggcctgaaa taacctctga aagaggaact tggttaggta ccttctgagg
     2821 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     2881 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     2941 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     3001 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     3061 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     3121 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctgtt
     3181 aacagcttgg cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     3241 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     3301 cgcaccgatc gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg
     3361 tattttctcc ttacgcatct gtgcggtatt tcacaccgca tacgtcaaag caaccatagt
     3421 acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg
     3481 ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca
     3541 cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
     3601 gtgctttacg gcacctcgac cccaaaaaac ttgatttggg tgatggttca cgtagtgggc
     3661 catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc tttaatagtg
     3721 gactcttgtt ccaaactgga acaacactca accctatctc gggctattct tttgatttat
     3781 aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa caaaaattta
     3841 acgcgaattt taacaaaata ttaacgttta caattttatg gtgcactctc agtacaatct
     3901 gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct gacgcgccct
     3961 gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct
     4021 gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gagacgaaag ggcctcgtga
     4081 tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg tcaggtggca
     4141 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     4201 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4261 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     4321 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     4381 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     4441 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     4501 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     4561 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     4621 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     4681 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     4741 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     4801 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     4861 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     4921 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     4981 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     5041 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5101 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     5161 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     5221 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5281 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5341 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5401 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     5461 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5521 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5581 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5641 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5701 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5761 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5821 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5881 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5941 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     6001 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gt
//
