LOCUS       sequence-139461-        8431 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 8431)
  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 8431)
  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 RagB 54L
FEATURES             Location/Qualifiers
     source          1..8431
                     /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    1384..1501
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1550..2060
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1979..1997
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2082..2681
                     /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(2082..2101)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2578..2598
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2809..3042
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3114..3235
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3151..3170)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3205..3224
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3275..3410
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3430..3449)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3431..3449)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3459..3476)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3459..3475)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3468..3490)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3617..4072
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3704..3723)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3914..3935
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4098..4202
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4203..4271,4272..5063)
                     /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(4421..4440)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5234..5822
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5723..5742
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5976..5993
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6110..6131
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6146..6151,6152..6169,6170..6176)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6184..6200
                     /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     6189..6211
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6208..6224
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6208..6224
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6243..6263
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6245..6263
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6291..6517
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6518..6698
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6745..6870
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7363..7596
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7781..7825
                     /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     8123..8143
                     /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 cgatggaaga
      301 atctgactct gagaaaacga cggagaaaga aaatctgggg ccgagaatgg atccaccact
      361 aggggaaccg gaaggatcgc ttgggtgggt gctaccaaat acagccatga agaaaaaggt
      421 gctgttgatg ggtaaaagtg ggtctggtaa gctcagcatg aggtctatta tctttgcaaa
      481 ttatattgcc agagacacac gtcgccttgg cgcaacaatt gatgtagaac attctcatgt
      541 tcgatttctg ggaaacctgg tattgaacct gtgggattgt ggtgggcaag acaccttcat
      601 ggaaaattat ttcactagcc aacgggacaa catcttccga aatgtggagg ttctgattta
      661 tgtctttgat gtggagagcc gcgaactgga aaaggacatg cactattacc aatcatgcct
      721 ggaggccatt ctgcagaatt ctccagatgc caaaatattt tgcttggtac acaaaatgga
      781 tctggtacag gaggatcaac gggacctgat ttttaaagag cgagaagaag atttgaggcg
      841 tttgtctcgc ccattggaat gttcttgttt ccgaacatct atctgggatg aaaccctcta
      901 taaggcttgg tccagcatag tttatcagct gattcccaat gttcagcagc tggaaatgaa
      961 cctaaggaat tttgctgaaa ttatcgaagc tgatgaagta cttctttttg agagagctac
     1021 ttttctggta atttctcact atcagtgtaa agagcagcgt gatgcccata gatttgagaa
     1081 aataagcaac attattaagc agttcaagct gagctgcagc aagctggctg cctctttcca
     1141 gagtatggaa gtcaggaact ctaacttcgc tgctttcatt gacatcttta catccaacac
     1201 ttatgtgatg gttgtgatgt ctgatccgtc cattccttct gcagctactc tgatcaacat
     1261 ccgcaatgcc aggaaacact ttgaaaagct ggaaagagtg gatggaccaa agcagtgtct
     1321 tctcatgcgc taagatatct tcgaattctc gacctcgaga caaatggcag tattcatcca
     1381 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     1441 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     1501 tcgggtttat tacagggaca gcagagatcc actttggccg cggctcgagg gggttggggt
     1561 tgcgcctttt ccaaggcagc cctgggtttg cgcagggacg cggctgctct gggcgtggtt
     1621 ccgggaaacg cagcggcgcc gaccctgggt ctcgcacatt cttcacgtcc gttcgcagcg
     1681 tcacccggat cttcgccgct acccttgtgg gccccccggc gacgcttcct gctccgcccc
     1741 taagtcggga aggttccttg cggttcgcgg cgtgccggac gtgacaaacg gaagccgcac
     1801 gtctcactag taccctcgca gacggacagc gccagggagc aatggcagcg cgccgaccgc
     1861 gatgggctgt ggccaatagc ggctgctcag cagggcgcgc cgagagcagc ggccgggaag
     1921 gggcggtgcg ggaggcgggg tgtggggcgg tagtgtgggc cctgttcctg cccgcgcggt
     1981 gttccgcatt ctgcaagcct ccggagcgca cgtcggcagt cggctccctc gttgaccgaa
     2041 tcaccgacct ctctccccag ggggatccac cggagcttac catgaccgag tacaagccca
     2101 cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt acgcaccctc gccgccgcgt
     2161 tcgccgacta ccccgccacg cgccacaccg tcgatccgga ccgccacatc gagcgggtca
     2221 ccgagctgca agaactcttc ctcacgcgcg tcgggctcga catcggcaag gtgtgggtcg
     2281 cggacgacgg cgccgcggtg gcggtctgga ccacgccgga gagcgtcgaa gcgggggcgg
     2341 tgttcgccga gatcggcccg cgcatggccg agttgagcgg ttcccggctg gccgcgcagc
     2401 aacagatgga aggcctcctg gcgccgcacc ggcccaagga gcccgcgtgg ttcctggcca
     2461 ccgtcggcgt ctcgcccgac caccagggca agggtctggg cagcgccgtc gtgctccccg
     2521 gagtggaggc ggccgagcgc gccggggtgc ccgccttcct ggagacctcc gcgccccgca
     2581 acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc cgacgtcgag gtgcccgaag
     2641 gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg acgcccgccc cacgacccgc
     2701 agcgcccgac cgaaaggagc gcacgacccc atgcatcggt acctttaaga ccaatgactt
     2761 acaaggcagc tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa
     2821 ttcactccca acgaagacaa gatctgcttt ttgcttgtac tgggtctctc tggttagacc
     2881 agatctgagc ctgggagctc tctggctaac tagggaaccc actgcttaag cctcaataaa
     2941 gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga
     3001 gatccctcag acccttttag tcagtgtgga aaatctctag cagtagtagt tcatgtcatc
     3061 ttattattca gtatttataa cttgcaaaga aatgaatatc agagagtgag aggaacttgt
     3121 ttattgcagc ttataatggt tacaaataaa gcaatagcat cacaaatttc acaaataaag
     3181 catttttttc actgcattct agttgtggtt tgtccaaact catcaatgta tcttatcatg
     3241 tctggctcta gctatcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     3301 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     3361 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc tagggacgta
     3421 cccaattcgc cctatagtga gtcgtattac gcgcgctcac tggccgtcgt tttacaacgt
     3481 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     3541 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     3601 ctgaatggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     3661 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     3721 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     3781 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     3841 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     3901 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     3961 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     4021 atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat ttaggtggca
     4081 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     4141 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     4201 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     4261 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     4321 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     4381 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     4441 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     4501 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     4561 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     4621 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     4681 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     4741 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     4801 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     4861 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     4921 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     4981 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     5041 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     5101 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     5161 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     5221 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     5281 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     5341 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     5401 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     5461 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     5521 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     5581 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     5641 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     5701 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     5761 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     5821 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5881 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     5941 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     6001 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     6061 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     6121 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     6181 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     6241 gcgcaattaa ccctcactaa agggaacaaa agctggagct gcaagcttaa tgtagtctta
     6301 tgcaatactc ttgtagtctt gcaacatggt aacgatgagt tagcaacatg ccttacaagg
     6361 agagaaaaag caccgtgcat gccgattggt ggaagtaagg tggtacgatc gtgccttatt
     6421 aggaaggcaa cagacgggtc tgacatggat tggacgaacc actgaattgc cgcattgcag
     6481 agatattgta tttaagtgcc tagctcgata cataaacggg tctctctggt tagaccagat
     6541 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt
     6601 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc
     6661 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg
     6721 aaagcgaaag ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc
     6781 acggcaagag gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc
     6841 tagaaggaga gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga
     6901 tgggaaaaaa ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta
     6961 tgggcaagca gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa
     7021 ggctgtagac aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt
     7081 agatcattat ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa
     7141 gacaccaagg aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca
     7201 cagcaagcgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     7261 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     7321 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     7381 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     7441 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     7501 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     7561 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     7621 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     7681 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     7741 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     7801 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     7861 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     7921 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     7981 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     8041 cagagacaga tccattcgat tagtgaacgg atctcgacgg tatcgatcac gagactagcc
     8101 tcgagcggcc gcccccttca ccgagggcct atttcccatg attccttcat atttgcatat
     8161 acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt
     8221 agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt
     8281 atgttttaaa atggactatc atatgccaag tacgccccct attgacgtca atgacggtaa
     8341 atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
     8401 catctacgta ttagtcatcg ctattaccat g
//
