LOCUS       sequence-235320-        8593 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 8593)
  AUTHORS   Sancak Y, Peterson TR, Shaul YD, Lindquist RA, Thoreen CC, Bar-Peled
            L, Sabatini DM
  TITLE     The Rag GTPases bind raptor and mediate amino acid signaling to 
            mTORC1.
  JOURNAL   Science. 2008 Jun 13. 320(5882):1496-501.
  PUBMED    18497260
REFERENCE   2  (bases 1 to 8593)
  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: "Science. 
            2008 Jun 13. 320(5882):1496-501."
COMMENT     Sequence Label: Flag pLJM1 RagD wt
FEATURES             Location/Qualifiers
     source          1..8593
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             245..268
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    1546..1663
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1712..2222
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2141..2159
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2244..2843
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(2244..2263)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2740..2760
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2971..3204
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3276..3397
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3313..3332)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3367..3386
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3437..3572
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3592..3611)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3593..3611)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3621..3638)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3621..3637)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3630..3652)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3779..4234
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3866..3885)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4076..4097
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4260..4364
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4365..4433,4434..5225)
                     /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(4583..4602)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5396..5984
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5885..5904
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6138..6155
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6272..6293
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6308..6313,6314..6331,6332..6338)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6346..6362
                     /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     6351..6373
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6370..6386
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6370..6386
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6405..6425
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6407..6425
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6453..6679
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6680..6860
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6907..7032
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7525..7758
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7943..7987
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     primer_bind     8285..8305
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
      241 tatggactac aaggatgacg atgacaaggg tggtggtggt ggtgcgtcga cgatgagcca
      301 ggtgctgggg aagccgcagc cgcaggacga ggacgacgcg gaggaggagg aggaggagga
      361 tgagctggtg gggctagcgg actacggaga cgggcccgac tcctccgacg ccgatccgga
      421 cagcggcaca gaggagggag ttctggactt cagtgacccc ttcagcactg aagtgaagcc
      481 gagaatcctg ctcatgggcc tgaggagaag cggcaagtcg tctattcaga aagttgtctt
      541 tcacaaaatg tctcccaacg aaactctgtt cttggagagc actaataaga tatgccggga
      601 agatgtttcc aacagctcct ttgtcaattt tcagatttgg gacttcccag gacagattga
      661 cttttttgac cctacatttg actatgagat gatcttccgg ggaacaggag cactgatatt
      721 tgtcattgac tcacaggatg attacatgga agccctggcc aggctccacc tcacggtgac
      781 cagggcctac aaagtgaata ctgacatcaa cttcgaggtg tttattcata aagtggatgg
      841 tctgtcagat gaccacaaaa ttgaaaccca aagagatatt caccagaggg caaacgatga
      901 ccttgcagat gctggattag aaaaaattca cctcagcttt tatctgacaa gcatatatga
      961 tcattcaata tttgaagctt ttagcaaagt tgttcagaaa ctgattccac aactcccaac
     1021 tctggagaat ttgctgaaca tctttatctc aaattctgga attgaaaagg catttctatt
     1081 tgatgtggtc agtaaaattt atattgcaac tgatagtact ccggtggata tgcaaaccta
     1141 tgagctctgc tgtgatatga tagatgtggt tattgacatc tcttgtattt atggtctcaa
     1201 agaagatgga gcaggaaccc cctatgacaa ggaatccaca gccatcataa agcttaataa
     1261 tacaaccgtg ctttatttaa aagaggtgac aaagttcctg gctctcgttt gctttgtcag
     1321 agaggaaagc tttgaaagaa aagggctaat tgactataat tttcattgct tccggaaggc
     1381 cattcatgaa gtttttgagg tgagaatgaa agtagtaaaa tctcgaaagg ttcagaatcg
     1441 gctgcagaag aaaaagagag ccacccctaa tgggacccct agagtgctgc tgtaggatat
     1501 cttcgaattc tcgacctcga gacaaatggc agtattcatc cacaatttta aaagaaaagg
     1561 ggggattggg gggtacagtg caggggaaag aatagtagac ataatagcaa cagacataca
     1621 aactaaagaa ttacaaaaac aaattacaaa aattcaaaat tttcgggttt attacaggga
     1681 cagcagagat ccactttggc cgcggctcga gggggttggg gttgcgcctt ttccaaggca
     1741 gccctgggtt tgcgcaggga cgcggctgct ctgggcgtgg ttccgggaaa cgcagcggcg
     1801 ccgaccctgg gtctcgcaca ttcttcacgt ccgttcgcag cgtcacccgg atcttcgccg
     1861 ctacccttgt gggccccccg gcgacgcttc ctgctccgcc cctaagtcgg gaaggttcct
     1921 tgcggttcgc ggcgtgccgg acgtgacaaa cggaagccgc acgtctcact agtaccctcg
     1981 cagacggaca gcgccaggga gcaatggcag cgcgccgacc gcgatgggct gtggccaata
     2041 gcggctgctc agcagggcgc gccgagagca gcggccggga aggggcggtg cgggaggcgg
     2101 ggtgtggggc ggtagtgtgg gccctgttcc tgcccgcgcg gtgttccgca ttctgcaagc
     2161 ctccggagcg cacgtcggca gtcggctccc tcgttgaccg aatcaccgac ctctctcccc
     2221 agggggatcc accggagctt accatgaccg agtacaagcc cacggtgcgc ctcgccaccc
     2281 gcgacgacgt ccccagggcc gtacgcaccc tcgccgccgc gttcgccgac taccccgcca
     2341 cgcgccacac cgtcgatccg gaccgccaca tcgagcgggt caccgagctg caagaactct
     2401 tcctcacgcg cgtcgggctc gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg
     2461 tggcggtctg gaccacgccg gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc
     2521 cgcgcatggc cgagttgagc ggttcccggc tggccgcgca gcaacagatg gaaggcctcc
     2581 tggcgccgca ccggcccaag gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg
     2641 accaccaggg caagggtctg ggcagcgccg tcgtgctccc cggagtggag gcggccgagc
     2701 gcgccggggt gcccgccttc ctggagacct ccgcgccccg caacctcccc ttctacgagc
     2761 ggctcggctt caccgtcacc gccgacgtcg aggtgcccga aggaccgcgc acctggtgca
     2821 tgacccgcaa gcccggtgcc tgacgcccgc cccacgaccc gcagcgcccg accgaaagga
     2881 gcgcacgacc ccatgcatcg gtacctttaa gaccaatgac ttacaaggca gctgtagatc
     2941 ttagccactt tttaaaagaa aaggggggac tggaagggct aattcactcc caacgaagac
     3001 aagatctgct ttttgcttgt actgggtctc tctggttaga ccagatctga gcctgggagc
     3061 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     3121 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     3181 agtcagtgtg gaaaatctct agcagtagta gttcatgtca tcttattatt cagtatttat
     3241 aacttgcaaa gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg
     3301 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     3361 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc
     3421 gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca
     3481 tggctgacta atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt
     3541 ccagaagtag tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt
     3601 gagtcgtatt acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct
     3661 ggcgttaccc aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc
     3721 gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac
     3781 gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct
     3841 acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg
     3901 ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt
     3961 gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca
     4021 tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga
     4081 ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa
     4141 gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac
     4201 gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc
     4261 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     4321 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt
     4381 tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag
     4441 aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg
     4501 aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa
     4561 tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc
     4621 aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag
     4681 tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa
     4741 ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc
     4801 taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg
     4861 agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa
     4921 caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg caacaattaa
     4981 tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg
     5041 gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag
     5101 cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg
     5161 caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt
     5221 ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt
     5281 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5341 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5401 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5461 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5521 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     5581 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5641 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5701 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5761 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5821 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5881 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5941 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6001 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6061 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     6121 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca
     6181 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     6241 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     6301 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     6361 aatttcacac aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact
     6421 aaagggaaca aaagctggag ctgcaagctt aatgtagtct tatgcaatac tcttgtagtc
     6481 ttgcaacatg gtaacgatga gttagcaaca tgccttacaa ggagagaaaa agcaccgtgc
     6541 atgccgattg gtggaagtaa ggtggtacga tcgtgcctta ttaggaaggc aacagacggg
     6601 tctgacatgg attggacgaa ccactgaatt gccgcattgc agagatattg tatttaagtg
     6661 cctagctcga tacataaacg ggtctctctg gttagaccag atctgagcct gggagctctc
     6721 tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt
     6781 agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc
     6841 agtgtggaaa atctctagca gtggcgcccg aacagggact tgaaagcgaa agggaaacca
     6901 gaggagctct ctcgacgcag gactcggctt gctgaagcgc gcacggcaag aggcgagggg
     6961 cggcgactgg tgagtacgcc aaaaattttg actagcggag gctagaagga gagagatggg
     7021 tgcgagagcg tcagtattaa gcgggggaga attagatcgc gatgggaaaa aattcggtta
     7081 aggccagggg gaaagaaaaa atataaatta aaacatatag tatgggcaag cagggagcta
     7141 gaacgattcg cagttaatcc tggcctgtta gaaacatcag aaggctgtag acaaatactg
     7201 ggacagctac aaccatccct tcagacagga tcagaagaac ttagatcatt atataataca
     7261 gtagcaaccc tctattgtgt gcatcaaagg atagagataa aagacaccaa ggaagcttta
     7321 gacaagatag aggaagagca aaacaaaagt aagaccaccg cacagcaagc ggccgctgat
     7381 cttcagacct ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa
     7441 agtagtaaaa attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca
     7501 gagagaaaaa agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg
     7561 aagcactatg ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg
     7621 tatagtgcag cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca
     7681 actcacagtc tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct
     7741 aaaggatcaa cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc
     7801 tgtgccttgg aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac
     7861 ctggatggag tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga
     7921 agaatcgcaa aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc
     7981 aagtttgtgg aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat
     8041 gatagtagga ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag
     8101 agttaggcag ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc
     8161 cgacaggccc gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg
     8221 attagtgaac ggatctcgac ggtatcgatc acgagactag cctcgagcgg ccgccccctt
     8281 caccgagggc ctatttccca tgattccttc atatttgcat atacgataca aggctgttag
     8341 agagataatt ggaattaatt tgactgtaaa cacaaagata ttagtacaaa atacgtgacg
     8401 tagaaagtaa taatttcttg ggtagtttgc agttttaaaa ttatgtttta aaatggacta
     8461 tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg cctggcatta
     8521 tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg tattagtcat
     8581 cgctattacc atg
//
