LOCUS       sequence-176302-        7987 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  3rd generation lentiviral vector for 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: pLJC2-Rap2A-3xFLAG
FEATURES             Location/Qualifiers
     source          1..7987
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      1..589
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     490..509
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     743..760
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    877..898
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(913..918,919..936,937..943)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    951..967
                     /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     956..978
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     975..991
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     975..991
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     1010..1030
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1012..1030
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        1058..1284
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             1285..1465
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1512..1637
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2130..2363
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2548..2592
                     /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     2890..2910
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     promoter        3199..3402
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     3353..3373
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     CDS             4025..4090
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     misc_feature    4138..4255
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        4304..4814
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     4733..4751
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             4836..5435
                     /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(4836..4855)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5332..5352
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             5563..5796
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    5868..5989
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5905..5924)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5959..5978
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      6029..6164
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(6184..6203)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(6185..6203)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(6213..6230)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(6213..6229)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6222..6244)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      6371..6826
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6458..6477)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6668..6689
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6852..6956
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6957..7025,7026..7817)
                     /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(7175..7194)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
       61 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
      121 cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag gccaccactt
      181 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
      241 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
      301 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
      361 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
      421 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
      481 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
      541 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
      601 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
      661 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
      721 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcccaat
      781 acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc acgacaggtt
      841 tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta
      901 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
      961 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
     1021 tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg
     1081 tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac
     1141 cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag
     1201 acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt
     1261 aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag
     1321 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
     1381 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     1441 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
     1501 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
     1561 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
     1621 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
     1681 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
     1741 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
     1801 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
     1861 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     1921 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcagcag
     1981 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     2041 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     2101 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     2161 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     2221 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     2281 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     2341 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     2401 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     2461 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     2521 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     2581 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     2641 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     2701 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     2761 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     2821 attcgattag tgaacggatc tcgacggtat cgatcacgag actagcctcg agcagcagcc
     2881 cccttcaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     2941 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
     3001 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     3061 gactatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
     3121 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
     3181 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
     3241 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
     3301 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
     3361 ggcggtaggc gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag
     3421 atccgctagc gctaccggtt taattaaagg gacgatgcgc gagtacaaag tggtggtgct
     3481 gggctcgggc ggggtaggca aatccgccct gaccgtgcag ttcgtgaccg gcaccttcat
     3541 cgagaaatac gaccccacca tcgaggactt ctaccgcaag gagatcgagg tggattcgtc
     3601 gccgtcggtg ctggagatcc tggacacggc gggcaccgag cagttcgcgt ccatgcggga
     3661 cctgtacatc aagaacggcc agggcttcat cctcgtctac agcctcgtca accagcagag
     3721 cttccaggac atcaagccca tgcgggacca gatcatccgc gtgaagcggt atgagaaagt
     3781 gccagtcatc ttggttggga acaaagtgga cctggaaagt gagagagaag tatcgtccag
     3841 cgaaggcaga gcccttgctg aagagtgggg ctgccccttt atggaaactt ccgctaagag
     3901 taaaacaatg gtggacgaac tctttgcaga aattgtgagg cagatgaact atgctgctca
     3961 gcctgacaaa gatgacccat gctgttctgc atgtaacata caaggaggca gcgcggccgc
     4021 aggcgactac aaagaccatg acggtgatta taaagatcat gacattgatt acaaggatga
     4081 cgatgacaag taataagaat tctcgacctc gagacaaatg gcagtattca tccacaattt
     4141 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     4201 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     4261 ttattacagg gacagcagag atccactttg gccgcggctc gagggggttg gggttgcgcc
     4321 ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt ggttccggga
     4381 aacgcagcgg cgccgaccct gggtctcgca cattcttcac gtccgttcgc agcgtcaccc
     4441 ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg cccctaagtc
     4501 gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca
     4561 ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga ccgcgatggg
     4621 ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg gaaggggcgg
     4681 tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg
     4741 cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg
     4801 acctctctcc ccagggggat ccaccggagc ttaccatgac cgagtacaag cccacggtgc
     4861 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg
     4921 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc
     4981 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg
     5041 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg
     5101 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga
     5161 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg
     5221 gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg
     5281 aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc
     5341 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc
     5401 gcacctggtg catgacccgc aagcccggtg cctgacgccc gccccacgac ccgcagcgcc
     5461 cgaccgaaag gagcgcacga ccccatgcat cggtaccttt aagaccaatg acttacaagg
     5521 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
     5581 cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct
     5641 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     5701 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     5761 tcagaccctt ttagtcagtg tggaaaatct ctagcagtag tagttcatgt catcttatta
     5821 ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg
     5881 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     5941 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc
     6001 tctagctatc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     6061 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     6121 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggga cgtacccaat
     6181 tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac
     6241 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     6301 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     6361 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     6421 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     6481 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     6541 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     6601 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     6661 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     6721 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     6781 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     6841 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     6901 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     6961 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     7021 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     7081 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     7141 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     7201 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     7261 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     7321 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     7381 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     7441 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     7501 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     7561 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     7621 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     7681 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     7741 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     7801 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     7861 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     7921 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     7981 gatcttc
//
