LOCUS       sequence-176301-        7987 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 7987)
  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 7987)
  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: pLJM1-Rap2A-3xFLAG
FEATURES             Location/Qualifiers
     source          1..7987
                     /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"
     misc_feature    940..1057
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        1106..1616
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     1535..1553
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             1638..2237
                     /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(1638..1657)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2134..2154
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             2365..2598
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    2670..2791
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2707..2726)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2761..2780
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      2831..2966
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(2986..3005)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2987..3005)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3015..3032)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(3015..3031)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3024..3046)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      3173..3628
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3260..3279)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3470..3491
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3654..3758
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3759..3827,3828..4619)
                     /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(3977..3996)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4790..5378
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5279..5298
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5532..5549
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5666..5687
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5702..5707,5708..5725,5726..5732)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5740..5756
                     /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     5745..5767
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5764..5780
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5764..5780
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     5799..5819
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        5801..5819
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        5847..6073
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             6074..6254
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    6301..6426
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    6919..7152
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             7337..7381
                     /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     7679..7699
                     /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 agtaataaga
      901 attctcgacc tcgagacaaa tggcagtatt catccacaat tttaaaagaa aaggggggat
      961 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagaca tacaaactaa
     1021 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattaca gggacagcag
     1081 agatccactt tggccgcggc tcgagggggt tggggttgcg ccttttccaa ggcagccctg
     1141 ggtttgcgca gggacgcggc tgctctgggc gtggttccgg gaaacgcagc ggcgccgacc
     1201 ctgggtctcg cacattcttc acgtccgttc gcagcgtcac ccggatcttc gccgctaccc
     1261 ttgtgggccc cccggcgacg cttcctgctc cgcccctaag tcgggaaggt tccttgcggt
     1321 tcgcggcgtg ccggacgtga caaacggaag ccgcacgtct cactagtacc ctcgcagacg
     1381 gacagcgcca gggagcaatg gcagcgcgcc gaccgcgatg ggctgtggcc aatagcggct
     1441 gctcagcagg gcgcgccgag agcagcggcc gggaaggggc ggtgcgggag gcggggtgtg
     1501 gggcggtagt gtgggccctg ttcctgcccg cgcggtgttc cgcattctgc aagcctccgg
     1561 agcgcacgtc ggcagtcggc tccctcgttg accgaatcac cgacctctct ccccaggggg
     1621 atccaccgga gcttaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
     1681 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
     1741 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
     1801 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
     1861 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
     1921 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     1981 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     2041 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     2101 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     2161 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     2221 gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac
     2281 gaccccatgc atcggtacct ttaagaccaa tgacttacaa ggcagctgta gatcttagcc
     2341 actttttaaa agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc
     2401 tgctttttgc ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     2461 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     2521 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     2581 tgtggaaaat ctctagcagt agtagttcat gtcatcttat tattcagtat ttataacttg
     2641 caaagaaatg aatatcagag agtgagagga acttgtttat tgcagcttat aatggttaca
     2701 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     2761 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gctctagcta tcccgcccct
     2821 aactccgccc atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg
     2881 actaattttt tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa
     2941 gtagtgagga ggcttttttg gaggcctagg gacgtaccca attcgcccta tagtgagtcg
     3001 tattacgcgc gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt
     3061 acccaactta atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag
     3121 gcccgcaccg atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc
     3181 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     3241 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     3301 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     3361 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
     3421 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg
     3481 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt
     3541 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat
     3601 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa
     3661 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     3721 cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg
     3781 tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc
     3841 tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg
     3901 atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga
     3961 gcacttttaa agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc
     4021 aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag
     4081 aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga
     4141 gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg
     4201 cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga
     4261 atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt
     4321 tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact
     4381 ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt
     4441 ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg
     4501 ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta
     4561 tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac
     4621 tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat ttttaattta
     4681 aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt
     4741 tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt
     4801 tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt
     4861 gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc
     4921 agataccaaa tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg
     4981 tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg
     5041 ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt
     5101 cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac
     5161 tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg
     5221 acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg
     5281 gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat
     5341 ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt
     5401 tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg
     5461 attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa
     5521 cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc
     5581 ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga
     5641 aagcgggcag tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg
     5701 ctttacactt tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc
     5761 acacaggaaa cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg
     5821 aacaaaagct ggagctgcaa gcttaatgta gtcttatgca atactcttgt agtcttgcaa
     5881 catggtaacg atgagttagc aacatgcctt acaaggagag aaaaagcacc gtgcatgccg
     5941 attggtggaa gtaaggtggt acgatcgtgc cttattagga aggcaacaga cgggtctgac
     6001 atggattgga cgaaccactg aattgccgca ttgcagagat attgtattta agtgcctagc
     6061 tcgatacata aacgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta
     6121 actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg
     6181 tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg
     6241 gaaaatctct agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag
     6301 ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga
     6361 ctggtgagta cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag
     6421 agcgtcagta ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca
     6481 gggggaaaga aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga
     6541 ttcgcagtta atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag
     6601 ctacaaccat cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca
     6661 accctctatt gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag
     6721 atagaggaag agcaaaacaa aagtaagacc accgcacagc aagcggccgc tgatcttcag
     6781 acctggagga ggagatatga gggacaattg gagaagtgaa ttatataaat ataaagtagt
     6841 aaaaattgaa ccattaggag tagcacccac caaggcaaag agaagagtgg tgcagagaga
     6901 aaaaagagca gtgggaatag gagctttgtt ccttgggttc ttgggagcag caggaagcac
     6961 tatgggcgca gcgtcaatga cgctgacggt acaggccaga caattattgt ctggtatagt
     7021 gcagcagcag aacaatttgc tgagggctat tgaggcgcaa cagcatctgt tgcaactcac
     7081 agtctggggc atcaagcagc tccaggcaag aatcctggct gtggaaagat acctaaagga
     7141 tcaacagctc ctggggattt ggggttgctc tggaaaactc atttgcacca ctgctgtgcc
     7201 ttggaatgct agttggagta ataaatctct ggaacagatt tggaatcaca cgacctggat
     7261 ggagtgggac agagaaatta acaattacac aagcttaata cactccttaa ttgaagaatc
     7321 gcaaaaccag caagaaaaga atgaacaaga attattggaa ttagataaat gggcaagttt
     7381 gtggaattgg tttaacataa caaattggct gtggtatata aaattattca taatgatagt
     7441 aggaggcttg gtaggtttaa gaatagtttt tgctgtactt tctatagtga atagagttag
     7501 gcagggatat tcaccattat cgtttcagac ccacctccca accccgaggg gacccgacag
     7561 gcccgaagga atagaagaag aaggtggaga gagagacaga gacagatcca ttcgattagt
     7621 gaacggatct cgacggtatc gatcacgaga ctagcctcga gcggccgccc ccttcaccga
     7681 gggcctattt cccatgattc cttcatattt gcatatacga tacaaggctg ttagagagat
     7741 aattggaatt aatttgactg taaacacaaa gatattagta caaaatacgt gacgtagaaa
     7801 gtaataattt cttgggtagt ttgcagtttt aaaattatgt tttaaaatgg actatcatat
     7861 gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca
     7921 gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat
     7981 taccatg
//
