LOCUS       sequence-442288-        8671 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of C-terminally Flag-tagged R2Tg protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8671)
  AUTHORS   Chen Y, Luo S, Hu Y, Mao B, Wang X, Lu Z, Shan Q, Zhang J, Wang S, 
            Feng G, Wang C, Liang C, Tang N, Niu R, Wang J, Han J, Yang N, Wang 
            H, Zhou Q, Li W
  TITLE     All-RNA-mediated targeted gene integration in mammalian cells with 
            rationally engineered R2 retrotransposons.
  JOURNAL   Cell. 2024 Jul 1:S0092-8674(24)00694-9. doi: 
            10.1016/j.cell.2024.06.020.
  PUBMED    38981481
REFERENCE   2  (bases 1 to 8671)
  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: "Cell. 2024 
            Jul 1:S0092-8674(24)00694-9. doi: 10.1016/j.cell.2024.06.020."
COMMENT     Sequence Label: pCAG-R2Tg-3xFlag
FEATURES             Location/Qualifiers
     source          1..8671
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        8..387
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          665..1681
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1605..1627
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1689..1708
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        1742..1760
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1772..1792
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5975..5995
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6002..6067
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    6119..6240
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6156..6175)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6210..6229
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6283..6299)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6283..6299)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6296..6318)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6307..6323
                     /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)."
     promoter        complement(join(6331..6337,6338..6355,6356..6361))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6376..6397
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6514..6531)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6685..7273)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6765..6784)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7444..8235,8236..8304))
                     /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     8067..8086
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8305..8409)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8488..8507)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 acgcgttgac attgattatt gactagttat taatagtaat caattacggg gtcattagtt
       61 catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga
      121 ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca
      181 atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca
      241 gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg
      301 cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc
      361 tacgtattag tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc
      421 cccatctccc ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt
      481 gcagcgatgg gggcgggggg gggggggggg cgcgccrggg gggggggggg ggggsgrggg
      541 gsggggsggg gsgrggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      601 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
      661 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
      721 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
      781 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
      841 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggggtgc
      901 gtgcgtgtgt gtgtgcgtgg ggagcgccgc gtgcggctcc gcgctgcccg gcggctgtga
      961 gcgctgcggg cgcggcgcgg ggctttgtgc gctccgcagt gtgcgcgagg ggagcgcggc
     1021 cgggggcggt gccccgcggt gcgggggggg ctgcgagggg aacaaaggct gcgtgcgggg
     1081 tgtgtgcgtg ggggggtgag cagggggtgt gggcgcgtcg gtcgggctgc aaccccccct
     1141 gcacccccct ccccgagttg ctgagcacgg cccggcttcg ggtgcggggc tccgtacggg
     1201 gcgtggcgcg gggctcgccg tgccgggcgg ggggtggcgg caggkggggg tgccgggcgg
     1261 ggcggggccg ccycgggccg gggrgggctc gggggrgggg cgcggcggcc cccggagcgc
     1321 cggcggctgt cgaggcgcgg cgagccgcag ccattgcctt ttatggtaat cgtgcgagag
     1381 ggcgcaggga cttcctttgt cccaaatctg tgcggagccg aaatctggga ggcgccgccg
     1441 caccccctct agcgggcgcg gggcgaagcg gtgcggcgcc ggcaggaagg aaatgggcgg
     1501 ggagggcctt cgtgcgtcgc cgcgccgccg tccccttctc cctctccagc ctcggggctg
     1561 tccgcggggg gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg
     1621 tgtgaccggc ggctctagcg cctctgctaa ccatgttcat gccttcttct ttttcctaca
     1681 gctcctgggc aacgtgctgg ttattgtgct gtctcatcat tttggcaaag ctagtgaatt
     1741 ctaatacgac tcactatagg ccgccaccat gcccaagaag aagaggaagg ttcccgggat
     1801 ggccagctgc cccaagccag gcccacctgt gtctgccggc gccatgagcc tggaatctgg
     1861 actgacaaca cacagcgtgc tggccatcga gagaggaccc aacagcctgg ccaattccgg
     1921 ctctgatttc ggcggcggcg gcctgggcct gccactaaga ctgctgcgtg tatcagtggg
     1981 cacgcagacc agcagaagcg attgggtgga tctggtgagc tggtcccacc ctggccctac
     2041 aagcaagtcc cagcaggttg accttgtgag cctgttcccg aagcaccggg tggacctgct
     2101 atccaaaaat gatcaggtgg atctggtcgc gcaattcctg ccttcaaagt tccctcccaa
     2161 cctggctgaa aatgacctcg ccctgctggt gaacctggaa ttttacagaa gcgacctgca
     2221 cgtgtacgaa tgcgtgcact tcgctgccca ttgggagggc ctgagcggac tgccagaagt
     2281 ctacgaacag ctcgcccctc agccttgcgt gggagagacc ctgcatagct ccctgcctcg
     2341 ggatagcgag ctgttcgtgc ctgaggaggg ctctagcgaa aaggaatcgg aagatgcccc
     2401 caagaccagc ccccccaccc ctggcaaaca tggcctggag cagacaggcg aggagaaggt
     2461 gatggttacc gtccccgaca agaaccctcc ctgtccttgc tgcggcacca gagtgaacag
     2521 cgtgctgaat ctgatcgagc acctgaaggt gagccacggc aagcgtggag tttgtttccg
     2581 gtgcgccaag tgcggcaagg aaaacagcaa ctaccacagc gtggtgtgcc actttcctaa
     2641 gtgtagaggc cctgagacag aaaaggcccc tgccggagaa tggatttgtg aggtgtgcaa
     2701 ccgggatttt accaccaaga tcggcctggg ccagcacaaa cggctggccc accccgccgt
     2761 gagaaaccag gagaggatcg tggccagcca acctaaagag acaagcaaca gaggcgccca
     2821 caagcggtgc tggacgaagg aagaggaaga attgttgatc agactggaag cccagttcga
     2881 gggaaacaag aacatcaaca agctgattgc cgagcacatc accaccaaga ccgccaagca
     2941 gatcagcgac aagagaaggc tgttaagcag aaagcctgcc gaggagccca gagaggaacc
     3001 tggcacctgc caccacaccc ggagagccgc cgcttcactg cggaccgagc ccgagatgtc
     3061 tcaccacgcc caggccgagg acagagataa cggccccggc agacggcctc tccccggaag
     3121 ggctgccgcc ggcggaagaa ccatggatga gatcagaaga caccccgaca aaggaaatgg
     3181 ccagcagcgg cctaccaagc agaaatccga agaacagctg caggcttact acaagaagac
     3241 cctggaagag agactgagcg ccggagccct gaacaccttt cctagagcct tcaaacaggt
     3301 gatggaaggc cgggacataa agctggtcat caaccagaca gcccaggatt gcttcggctg
     3361 tctggagagc atctcccaga ttaggaccgc tacacgggat aagaaggata ccgtgacaag
     3421 agagaaacac cctaagaagc ccttccagaa gtggatgaaa gacagagcaa tcaagaaagg
     3481 caattacctg agattccaga gactgtttta cctggacaga ggcaagctgg ccaaaatcat
     3541 tctggacgac atcgagtgtc tgagttgcga catccctctg tccgagatct acagcgtgtt
     3601 caaaactcgg tgggaaacaa caggcagctt caagtctctg ggcgacttca agacatacgg
     3661 caaagccgat aacacggctt ttagagagct gatcaccgcc aaggagattg aaaagaacgt
     3721 gcaggagatg agcaagggca gcgcccctgg ccccgatggc atcaccctgg gagatgtggt
     3781 gaagatggac cctgagttca gcagaacgat ggagattttc aatctgtggc tgaccaccgg
     3841 caagattccc gacatggtgc ggggatgtcg gaccgtgctg atcccaaaga gctccaagcc
     3901 cgatagactg aaggacatca acaactggag acccatcacc atcggcagca tcctactcag
     3961 actgtttagc agaatcgtga ccgccagact gtccaaggct tgtcctctga atcctaggca
     4021 gagaggcttc atccgggctg ctggttgcag cgagaacctg aagctgttgc agaccatcat
     4081 ctggagcgcc aagagagagc acagacctct gggcgtggtg tttgtggaca tcgccaaagc
     4141 cttcgacacc gtgtcccacc agcatatcat ccacgccctg caacaaagag aggtggaccc
     4201 ccacatcgtg ggcctggtca gcaacatgta cgagaacatc agcacctaca tcacaaccaa
     4261 gcggaacacc cacaccgaca agatccagat ccgggtgggc gtgaagcagg gcgatcctat
     4321 gtcccctctt ctgttcaacc tggccatgga ccccctgttg tgcaagctgg aggagagcgg
     4381 caagggctac caccggggcc agagcagcat cacagccatg gcgttcgccg acgatttggt
     4441 gttgctgagc gattcttggg agaacatgaa caccaacatc agcatcctgg agaccttctg
     4501 caacctgacc ggcctgaaaa cccagggcca gaagtgccac ggcttctata tcaagcctac
     4561 aaaagacagc tacaccatca atgactgcgc cgcctggacc atcaacggca cacctctgaa
     4621 tatgattgac cctggcgaaa gcgagaagta cctgggcttg cagttcgacc cttggatcgg
     4681 aatcgccaga agcggcctga gcaccaagct cgacttctgg ttgcagagaa tcgaccaggc
     4741 accactgaag cccctgcaaa agacagacat cctgaagaca tacaccatcc ctcgcctaat
     4801 ctatatcgcc gaccactctg aggtgaaaac cgccctgctg gaaaccctgg accaaaagat
     4861 ccggacagcc gtgaaggaat ggctgcacct gccgccttgt acatgcgacg ctatcctgta
     4921 cagcagcacc agagatggcg ggctgggcat cactaagctg gccggcctca ttcctagcgt
     4981 gcaggccaga cggctgcata gaatcgcaca aagctctgat gacaccatga agtgctttat
     5041 ggagaaagag aaaatggaac agctgcacaa gaagctgtgg attcaggccg gtggagaccg
     5101 agaaaacatc ccatctatct gggaggctcc tcctagctct gagcctccta acaacgtgtc
     5161 tacaaacagc gagtgggagg cccctaccca gaaggacaag ttccccaagc catgtaactg
     5221 gcggaaaaac gagttcaaga agtggaccaa gctggccagc cagggccggg gcattgtgaa
     5281 cttcgagcgg gacaagatct ctaaccactg gattcagtac tatagaagaa tccctcaccg
     5341 gaagctgctg accgccctgc agctgagagc taatgtatac cccacaagag agttcctggc
     5401 cagaggaaga caggaccagt acatcaaagc ctgcagacac tgcgatgctg acatcgaaag
     5461 ctgtgcacat atcatcggca actgccccgt gacccaggac gcccgcataa agcgtcacaa
     5521 ctatatctgc gagcttctgc tggaagaagc caaaaagaaa gattgggtgg tgtttaagga
     5581 acctcacatc cgggacagca acaaggagct gtacaagcct gatctgatct tcgtcaagga
     5641 cgccagagct ctggttgtgg acgtgactgt gagatacgag gccgctaagt cctccctgga
     5701 agaggccgcc gccgagaagg tgagaaagta caaacacctg gaaaccgagg tgagacacct
     5761 gaccaacgcc aaagacgtga ccttcgtcgg tttccctctg ggcgctcgcg gcaagtggca
     5821 ccaggacaac ttcaagctcc tgactgaact gggactgtct aaaagcagac aggtgaagat
     5881 ggcagagaca ttttccaccg tggctctgtt ctcctccgtg gacatcgtcc acatgttcgc
     5941 cagcagagcc cgaaaaagca tggtgatggc tagcccaaag aagaagagga aagtcggatc
     6001 cgactacaaa gaccatgacg gtgattataa agatcatgac atcgattaca aggatgacga
     6061 tgacaagtaa tctagactgc agcgcgggga tctcatgctg gagttcttcg cccaccccaa
     6121 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     6181 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     6241 ttaattaacg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     6301 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     6361 agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     6421 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     6481 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     6541 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     6601 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     6661 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     6721 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     6781 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     6841 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     6901 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     6961 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     7021 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     7081 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     7141 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     7201 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     7261 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     7321 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     7381 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     7441 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     7501 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     7561 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     7621 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     7681 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     7741 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     7801 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     7861 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     7921 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     7981 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     8041 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     8101 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     8161 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     8221 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     8281 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     8341 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     8401 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatcgatc
     8461 tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat agttaagcca
     8521 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
     8581 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     8641 tgcgctgctt cgcgatgtac gggccagata t
//
