LOCUS       sequence-164357-        8329 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses human RagA from a CMV promoter for stable expression in 
            mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8329)
  AUTHORS   Efeyan A, Schweitzer LD, Bilate AM, Chang S, Kirak O, Lamming DW, 
            Sabatini DM
  TITLE     RagA, but not RagB, is essential for embryonic development and adult
            mice.
  JOURNAL   Dev Cell. 2014 May 12;29(3):321-9. doi: 
            10.1016/j.devcel.2014.03.017. Epub 2014 Apr 24.
  PUBMED    24768164
REFERENCE   2  (bases 1 to 8329)
  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"; doi: 
            "10.1016/j.devcel.2014.03.017"; journalName: "Dev Cell"; date: 
            "2014-05-12- 12"; volume: "29"; issue: "3"; pages: "321-9"
COMMENT     Sequence Label: pLJM1 RagA
FEATURES             Location/Qualifiers
     source          1..8329
                     /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"
     regulatory      372..381
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             381..404
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    1412..1529
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1578..2088
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2007..2025
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             2110..2709
                     /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(2110..2129)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2606..2626
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2837..3070
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    3142..3263
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3179..3198)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3233..3252
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      3303..3438
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(3458..3477)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3459..3477)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3487..3504)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3487..3503)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3496..3518)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3645..4100
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3732..3751)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3942..3963
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        4126..4230
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4231..4299,4300..5091)
                     /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(4449..4468)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5262..5850
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5751..5770
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6004..6021
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    6138..6159
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(6174..6179,6180..6197,6198..6204)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6212..6228
                     /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     6217..6239
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     6236..6252
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6236..6252
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     6271..6291
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        6273..6291
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        6319..6545
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6546..6726
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6773..6898
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    7391..7624
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7809..7853
                     /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     8151..8171
                     /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 tgccaccatg gactacaagg atgacgatga caagggtggt ggtggtggtg
      421 cgtcgacgat gccaaataca gccatgaaga aaaaggtgct gctgatgggg aagagcgggt
      481 cggggaagac cagcatgagg tcgataatct tcgccaatta cattgctcgc gacacccggc
      541 gcctgggggc caccattgac gtggaacact cccacgtccg attcctaggg aacctggtgc
      601 tgaacctgtg ggactgtggc ggtcaggaca ccttcatgga aaattacttc accagccagc
      661 gagacaatat cttccgtaac gtggaagttt tgatttacgt gtttgacgtg gagagccgcg
      721 aactggaaaa ggacatgcat tattaccagt cgtgtctgga ggccatcctc cagaactctc
      781 ctgacgccaa aatcttctgc ctggtgcaca aaatggatct ggttcaggag gatcagcgtg
      841 acctgatttt taaagagcga gaggaagacc tgaggcgtct gtctcgcccg ctggagtgtg
      901 cttgttttcg aacgtccatc tgggatgaga cgctctacaa agcctggtcc agcatcgtct
      961 accagctgat tcccaacgtt cagcagctgg agatgaacct caggaatttt gcccaaatca
     1021 ttgaggccga tgaagttctg ctgttcgaaa gagctacatt cttggttatt tcccactacc
     1081 agtgcaaaga gcagcgcgac gtccaccggt ttgagaagat cagcaacatc atcaaacagt
     1141 tcaagctgag ctgcagtaaa ttggccgctt ccttccagag catggaagtt aggaattcca
     1201 acttcgctgc tttcatcgac atcttcacct caaatacgta cgtgatggtg gtcatgtcag
     1261 atccgtcgat cccttctgcg gccactctga tcaacattcg caatgcccgg aaacactttg
     1321 agaagctgga gagagtggat ggccccaagc acagtctcct tatgcgttga caattctcga
     1381 cctcgagaca aatggcagta ttcatccaca attttaaaag aaaagggggg attggggggt
     1441 acagtgcagg ggaaagaata gtagacataa tagcaacaga catacaaact aaagaattac
     1501 aaaaacaaat tacaaaaatt caaaattttc gggtttatta cagggacagc agagatccac
     1561 tttggccgcg gctcgagggg gttggggttg cgccttttcc aaggcagccc tgggtttgcg
     1621 cagggacgcg gctgctctgg gcgtggttcc gggaaacgca gcggcgccga ccctgggtct
     1681 cgcacattct tcacgtccgt tcgcagcgtc acccggatct tcgccgctac ccttgtgggc
     1741 cccccggcga cgcttcctgc tccgccccta agtcgggaag gttccttgcg gttcgcggcg
     1801 tgccggacgt gacaaacgga agccgcacgt ctcactagta ccctcgcaga cggacagcgc
     1861 cagggagcaa tggcagcgcg ccgaccgcga tgggctgtgg ccaatagcgg ctgctcagca
     1921 gggcgcgccg agagcagcgg ccgggaaggg gcggtgcggg aggcggggtg tggggcggta
     1981 gtgtgggccc tgttcctgcc cgcgcggtgt tccgcattct gcaagcctcc ggagcgcacg
     2041 tcggcagtcg gctccctcgt tgaccgaatc accgacctct ctccccaggg ggatccaccg
     2101 gagcttacca tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc
     2161 agggccgtac gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc
     2221 gatccggacc gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc
     2281 gggctcgaca tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc
     2341 acgccggaga gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag
     2401 ttgagcggtt cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg
     2461 cccaaggagc ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag
     2521 ggtctgggca gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc
     2581 gccttcctgg agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc
     2641 gtcaccgccg acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc
     2701 ggtgcctgac gcccgcccca cgacccgcag cgcccgaccg aaaggagcgc acgaccccat
     2761 gcatcggtac ctttaagacc aatgacttac aaggcagctg tagatcttag ccacttttta
     2821 aaagaaaagg ggggactgga agggctaatt cactcccaac gaagacaaga tctgcttttt
     2881 gcttgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     2941 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     3001 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     3061 atctctagca gtagtagttc atgtcatctt attattcagt atttataact tgcaaagaaa
     3121 tgaatatcag agagtgagag gaacttgttt attgcagctt ataatggtta caaataaagc
     3181 aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg
     3241 tccaaactca tcaatgtatc ttatcatgtc tggctctagc tatcccgccc ctaactccgc
     3301 ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt
     3361 tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag aagtagtgag
     3421 gaggcttttt tggaggccta gggacgtacc caattcgccc tatagtgagt cgtattacgc
     3481 gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
     3541 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
     3601 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc cctgtagcgg
     3661 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
     3721 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     3781 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     3841 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac
     3901 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     3961 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat
     4021 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     4081 aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4141 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     4201 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     4261 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     4321 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     4381 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     4441 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     4501 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     4561 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     4621 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     4681 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     4741 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     4801 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     4861 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     4921 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     4981 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     5041 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     5101 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     5161 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     5221 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     5281 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     5341 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     5401 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     5461 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     5521 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     5581 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     5641 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     5701 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     5761 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     5821 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     5881 ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc tgattctgtg
     5941 gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg aacgaccgag
     6001 cgcagcgagt cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc gcctctcccc
     6061 gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg gaaagcgggc
     6121 agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca ggctttacac
     6181 tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt tcacacagga
     6241 aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag ggaacaaaag
     6301 ctggagctgc aagcttaatg tagtcttatg caatactctt gtagtcttgc aacatggtaa
     6361 cgatgagtta gcaacatgcc ttacaaggag agaaaaagca ccgtgcatgc cgattggtgg
     6421 aagtaaggtg gtacgatcgt gccttattag gaaggcaaca gacgggtctg acatggattg
     6481 gacgaaccac tgaattgccg cattgcagag atattgtatt taagtgccta gctcgataca
     6541 taaacgggtc tctctggtta gaccagatct gagcctggga gctctctggc taactaggga
     6601 acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc
     6661 tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct
     6721 ctagcagtgg cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg
     6781 acgcaggact cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag
     6841 tacgccaaaa attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag
     6901 tattaagcgg gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa
     6961 gaaaaaatat aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt
     7021 taatcctggc ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc
     7081 atcccttcag acaggatcag aagaacttag atcattatat aatacagtag caaccctcta
     7141 ttgtgtgcat caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga
     7201 agagcaaaac aaaagtaaga ccaccgcaca gcaagcggcc gctgatcttc agacctggag
     7261 gaggagatat gagggacaat tggagaagtg aattatataa atataaagta gtaaaaattg
     7321 aaccattagg agtagcaccc accaaggcaa agagaagagt ggtgcagaga gaaaaaagag
     7381 cagtgggaat aggagctttg ttccttgggt tcttgggagc agcaggaagc actatgggcg
     7441 cagcgtcaat gacgctgacg gtacaggcca gacaattatt gtctggtata gtgcagcagc
     7501 agaacaattt gctgagggct attgaggcgc aacagcatct gttgcaactc acagtctggg
     7561 gcatcaagca gctccaggca agaatcctgg ctgtggaaag atacctaaag gatcaacagc
     7621 tcctggggat ttggggttgc tctggaaaac tcatttgcac cactgctgtg ccttggaatg
     7681 ctagttggag taataaatct ctggaacaga tttggaatca cacgacctgg atggagtggg
     7741 acagagaaat taacaattac acaagcttaa tacactcctt aattgaagaa tcgcaaaacc
     7801 agcaagaaaa gaatgaacaa gaattattgg aattagataa atgggcaagt ttgtggaatt
     7861 ggtttaacat aacaaattgg ctgtggtata taaaattatt cataatgata gtaggaggct
     7921 tggtaggttt aagaatagtt tttgctgtac tttctatagt gaatagagtt aggcagggat
     7981 attcaccatt atcgtttcag acccacctcc caaccccgag gggacccgac aggcccgaag
     8041 gaatagaaga agaaggtgga gagagagaca gagacagatc cattcgatta gtgaacggat
     8101 ctcgacggta tcgatcacga gactagcctc gagcggccgc ccccttcacc gagggcctat
     8161 ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag ataattggaa
     8221 ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga aagtaataat
     8281 ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatca
//
