LOCUS       sequence-150768-        7942 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 7942)
  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 7942)
  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 Rap2A
FEATURES             Location/Qualifiers
     source          1..7942
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        131..334
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     285..305
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             375..398
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    1025..1142
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1191..1701
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1620..1638
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1723..2322
                     /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(1723..1742)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2219..2239
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2450..2683
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2755..2876
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2792..2811)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2846..2865
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2916..3051
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3071..3090)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3072..3090)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3100..3117)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3100..3116)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3109..3131)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3258..3713
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3345..3364)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3555..3576
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3739..3843
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3844..3912,3913..4704)
                     /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(4062..4081)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4875..5463
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5364..5383
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5617..5634
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5751..5772
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5787..5792,5793..5810,5811..5817)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5825..5841
                     /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     5830..5852
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5849..5865
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5849..5865
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5884..5904
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5886..5904
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5932..6158
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6159..6339
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6386..6511
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7004..7237
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7422..7466
                     /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     7764..7784
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
ORIGIN
        1 tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc
       61 ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat tagtcatcgc
      121 tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc
      181 acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa
      241 tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag
      301 gcgtgtacgg tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta
      361 gcgctaccgg tatggactac aaggatgacg atgacaaggg tggtggtggt ggtgcgtcga
      421 cgatgcgcga gtacaaagtg gtggtgctgg gctcgggcgg ggtaggcaaa tccgccctga
      481 ccgtgcagtt cgtgaccggc accttcatcg agaaatacga ccccaccatc gaggacttct
      541 accgcaagga gatcgaggtg gattcgtcgc cgtcggtgct ggagatcctg gacacggcgg
      601 gcaccgagca gttcgcgtcc atgcgggacc tgtacatcaa gaacggccag ggcttcatcc
      661 tcgtctacag cctcgtcaac cagcagagct tccaggacat caagcccatg cgggaccaga
      721 tcatccgcgt gaagcggtat gagaaagtgc cagtcatctt ggttgggaac aaagtggacc
      781 tggaaagtga gagagaagta tcgtccagcg aaggcagagc ccttgctgaa gagtggggct
      841 gcccctttat ggaaacttcc gctaagagta aaacaatggt ggacgaactc tttgcagaaa
      901 ttgtgaggca gatgaactat gctgctcagc ctgacaaaga tgacccatgc tgttctgcat
      961 gtaacataca ataggatatc ttcgaattct cgacctcgag acaaatggca gtattcatcc
     1021 acaattttaa aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca
     1081 taatagcaac agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt
     1141 ttcgggttta ttacagggac agcagagatc cactttggcc gcggctcgag ggggttgggg
     1201 ttgcgccttt tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt
     1261 tccgggaaac gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc
     1321 gtcacccgga tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc
     1381 ctaagtcggg aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca
     1441 cgtctcacta gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg
     1501 cgatgggctg tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa
     1561 ggggcggtgc gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg
     1621 tgttccgcat tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga
     1681 atcaccgacc tctctcccca gggggatcca ccggagctta ccatgaccga gtacaagccc
     1741 acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg
     1801 ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc
     1861 accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc
     1921 gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg
     1981 gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag
     2041 caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc
     2101 accgtcggcg tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc
     2161 ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc
     2221 aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa
     2281 ggaccgcgca cctggtgcat gacccgcaag cccggtgcct gacgcccgcc ccacgacccg
     2341 cagcgcccga ccgaaaggag cgcacgaccc catgcatcgg tacctttaag accaatgact
     2401 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
     2461 attcactccc aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac
     2521 cagatctgag cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa
     2581 agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag
     2641 agatccctca gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat
     2701 cttattattc agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg
     2761 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     2821 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     2881 gtctggctct agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt
     2941 ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg
     3001 cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt
     3061 acccaattcg ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg
     3121 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     3181 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     3241 cctgaatggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt
     3301 tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt
     3361 cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc
     3421 tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga
     3481 tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc
     3541 cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt
     3601 ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct
     3661 gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc
     3721 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     3781 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     3841 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     3901 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     3961 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     4021 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4081 tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     4141 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4201 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4261 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4321 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4381 atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4441 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     4501 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     4561 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4621 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     4681 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     4741 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4801 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4861 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4921 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4981 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     5041 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5101 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5161 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5221 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5281 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5341 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5401 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5461 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5521 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     5581 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     5641 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     5701 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt
     5761 tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt
     5821 ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag
     5881 cgcgcaatta accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt
     5941 atgcaatact cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag
     6001 gagagaaaaa gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat
     6061 taggaaggca acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca
     6121 gagatattgt atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga
     6181 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     6241 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     6301 ccctcagacc cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt
     6361 gaaagcgaaa gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg
     6421 cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg
     6481 ctagaaggag agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg
     6541 atgggaaaaa attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt
     6601 atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga
     6661 aggctgtaga caaatactgg gacagctaca accatccctt cagacaggat cagaagaact
     6721 tagatcatta tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa
     6781 agacaccaag gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc
     6841 acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa
     6901 gtgaattata taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg
     6961 caaagagaag agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg
     7021 ggttcttggg agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg
     7081 ccagacaatt attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg
     7141 cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc
     7201 tggctgtgga aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa
     7261 aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac
     7321 agatttggaa tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct
     7381 taatacactc cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat
     7441 tggaattaga taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt
     7501 atataaaatt attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg
     7561 tactttctat agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc
     7621 tcccaacccc gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag
     7681 acagagacag atccattcga ttagtgaacg gatctcgacg gtatcgatca cgagactagc
     7741 ctcgagcggc cgcccccttc accgagggcc tatttcccat gattccttca tatttgcata
     7801 tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat
     7861 tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat
     7921 tatgttttaa aatggactat ca
//
