LOCUS       sequence-176299-        7267 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of Rap2A in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7267)
  AUTHORS   Cantor JR, Abu-Remaileh M, Kanarek N, Freinkman E, Gao X, Louissaint
            A Jr, Lewis CA, Sabatini DM
  TITLE     Physiologic Medium Rewires Cellular Metabolism and Reveals Uric Acid
            as an Endogenous Inhibitor of UMP Synthase.
  JOURNAL   Cell. 2017 Apr 6;169(2):258-272.e17. doi: 
            10.1016/j.cell.2017.03.023.
  PUBMED    28388410
REFERENCE   2  (bases 1 to 7267)
  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.cell.2017.03"; journalName: "Cell"; date: "2017-04-6- 6";
            volume: "169"; issue: "2"; pages: "258-272.e17"
COMMENT     Sequence Label: pLJC1-Rap2A-3xFLAG
FEATURES             Location/Qualifiers
     source          1..7267
                     /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             827..892
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             918..1517
                     /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(918..937)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1414..1434
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1645..1878
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    1950..2071
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1987..2006)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2041..2060
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2111..2246
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2266..2285)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2267..2285)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2295..2312)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2295..2311)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2304..2326)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2453..2908
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2540..2559)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2750..2771
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        2934..3038
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3039..3107,3108..3899)
                     /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(3257..3276)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4070..4658
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4559..4578
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4812..4829
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4946..4967
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4982..4987,4988..5005,5006..5012)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5020..5036
                     /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     5025..5047
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5044..5060
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5044..5060
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5079..5099
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5081..5099
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5127..5353
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5354..5534
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5581..5706
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6199..6432
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6617..6661
                     /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     6959..6979
                     /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 tttaattaaa gggacgatgc gcgagtacaa agtggtggtg ctgggctcgg gcggggtagg
      301 caaatccgcc ctgaccgtgc agttcgtgac cggcaccttc atcgagaaat acgaccccac
      361 catcgaggac ttctaccgca aggagatcga ggtggattcg tcgccgtcgg tgctggagat
      421 cctggacacg gcgggcaccg agcagttcgc gtccatgcgg gacctgtaca tcaagaacgg
      481 ccagggcttc atcctcgtct acagcctcgt caaccagcag agcttccagg acatcaagcc
      541 catgcgggac cagatcatcc gcgtgaagcg gtatgagaaa gtgccagtca tcttggttgg
      601 gaacaaagtg gacctggaaa gtgagagaga agtatcgtcc agcgaaggca gagcccttgc
      661 tgaagagtgg ggctgcccct ttatggaaac ttccgctaag agtaaaacaa tggtggacga
      721 actctttgca gaaattgtga ggcagatgaa ctatgctgct cagcctgaca aagatgaccc
      781 atgctgttct gcatgtaaca tacaaggagg cagcgcggcc gcaggcgact acaaagacca
      841 tgacggtgat tataaagatc atgacattga ttacaaggat gacgatgaca agtaataagg
      901 atccaccgga gcttaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
      961 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     1021 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     1081 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     1141 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     1201 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     1261 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     1321 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     1381 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     1441 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     1501 gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac
     1561 gaccccatgc atcggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc
     1621 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc
     1681 tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     1741 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     1801 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     1861 tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg
     1921 caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca
     1981 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     2041 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct
     2101 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     2161 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     2221 gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg
     2281 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     2341 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     2401 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc
     2461 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     2521 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2581 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2641 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     2701 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     2761 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     2821 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     2881 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     2941 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3001 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3061 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3121 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3181 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     3241 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     3301 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     3361 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     3421 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     3481 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     3541 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     3601 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     3661 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     3721 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     3781 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     3841 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     3901 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     3961 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4021 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4081 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4141 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4201 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4261 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     4321 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     4381 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     4441 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     4501 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     4561 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     4621 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     4681 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     4741 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     4801 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     4861 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     4921 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     4981 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     5041 acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg
     5101 aacaaaagct ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa
     5161 catggtaacg atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatgccg
     5221 attggtggaa gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac
     5281 atggattgga cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc
     5341 tcgatacata aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     5401 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     5461 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     5521 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag
     5581 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga
     5641 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag
     5701 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca
     5761 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga
     5821 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
     5881 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
     5941 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
     6001 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcagcagc tgatcttcag
     6061 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
     6121 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
     6181 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
     6241 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
     6301 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
     6361 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
     6421 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
     6481 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
     6541 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
     6601 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
     6661 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
     6721 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag
     6781 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag
     6841 gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt
     6901 gaacggatct cgacggtatc gatcacgaga ctagcctcga gcagcagccc ccttcaccga
     6961 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
     7021 aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
     7081 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
     7141 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     7201 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     7261 taccatg
//
