LOCUS       sequence-442290-        9280 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of C-terminally Flag-tagged optimized R2Tg 
            protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9280)
  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 9280)
  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_opt-3xFlag
FEATURES             Location/Qualifiers
     source          1..9280
                     /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             6584..6604
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             6611..6676
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     polyA_signal    6728..6849
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6765..6784)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6819..6838
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6892..6908)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6892..6908)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6905..6927)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6916..6932
                     /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(6940..6946,6947..6964,6965..6970))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6985..7006
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7123..7140)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7294..7882)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7374..7393)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8053..8844,8845..8913))
                     /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     8676..8695
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8914..9018)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(9097..9116)
                     /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 cgcgccrggs gggggggggg ggggsgrggg
      541 ggggggsggg 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 gcctaagaga aaggtgtcca gcgccgaggg cgccgccaaa gaagagccta agcggagaag
     1861 cgccagactg agcgccaagc ctccagctaa agtggaagcc aagcccaaga aggccgctgc
     1921 caaggacaag agctctgata agaaagtcca gaccaaggga aaacggggcg ctaaaggcaa
     1981 gcaggccgag gtggccaacc aggagacaaa ggaagatctg cctgctgaga acggcgaaac
     2041 caagaccgag gaaagccccg ccagcgacga ggccggcgag aaggaggcca agtctgacgc
     2101 cagctgcccc aagccaggcc cacctgtgtc tgccggcgcc atgagcctgg aatctggact
     2161 gacaacacac agcgtgctgg ccatcgagag aggacccaac agcctggcca attccggctc
     2221 tgatttcggc ggcggcggcc tgggcctgcc actaagactg ctgcgtgtat cagtgggcac
     2281 gcagaccagc agaagcgatt gggtggatct ggtgagctgg tcccaccctg gccctacaag
     2341 caagtcccag caggttgacc ttgtgagcct gttcccgaag caccgggtgg acctgctatc
     2401 caaaaatgat caggtggatc tggtcgcgca attcctgcct tcaaagttcc ctcccaacct
     2461 ggctgaaaat gacctcgccc tgctggtgaa cctggaattt tacagaagcg acctgcacgt
     2521 gtacgaatgc gtgcacttcg ctgcccattg ggagggcctg agcggactgc cagaagtcta
     2581 cgaacagctc gcccctcagc cttgcgtggg agagaccctg catagctccc tgcctcggga
     2641 tagcgagctg ttcgtgcctg aggagggctc tagcgaaaag gaatcggaag atgcccccaa
     2701 gaccagcccc cccacccctg gcaaacatgg cctggagcag acaggcgagg agaaggtgat
     2761 ggttaccgtc cccgacaaga accctccctg tccttgctgc ggcaccagag tgaacagcgt
     2821 gctgaatctg atcgagcacc tgaaggtgag ccacggcaag cgtggagttt gtttccggtg
     2881 cgccaagtgc ggcaaggaaa acagcaacta ccacagcgtg gtgtgccact ttcctaagtg
     2941 tagaggccct gagacagaaa aggcccctgc cggagaatgg atttgtgagg tgtgcaaccg
     3001 ggattttacc accaagatcg gcctgggcca gcacaaacgg ctggcccacc ccgccgtgag
     3061 aaaccaggag aggatcgtgg ccagccaacc taaagagaca agcaacagag gcgcccacaa
     3121 gcggtgctgg acgaaggaag aggaagaatt gttgatcaga ctggaagccc agttcgaggg
     3181 aaacaagaac atcaacaagc tgattgccga gcacatcacc accaagaccg ccaagcagat
     3241 cagcgacaag agaaggctgt taagcagaaa gcctgccgag gagcccagag aggaacctgg
     3301 cacctgccac cacacccgga gagccgccgc ttcactgcgg accgagcccg agatgtctca
     3361 ccacgcccag gccgaggaca gagataacgg cggaggaatg cctaagagaa aggtgtccag
     3421 cgccgagggc gccgccaaag aagagcctaa gcggagaagc gccagactga gcgccaagcc
     3481 tccagctaaa gtggaagcca agcccaagaa ggccgctgcc aaggacaaga gctctgataa
     3541 gaaagtccag accaagggaa aacggggcgc taaaggcaag caggccgagg tggccaacca
     3601 ggagacaaag gaagatctgc ctgctgagaa cggcgaaacc aagaccgagg aaagccccgc
     3661 cagcgacgag gccggcgaga aggaggccaa gtctgacgga ggacccggca gacggcctct
     3721 ccccggaagg gctgccgccg gcggaagaac catggatgag atcagaagac accccgacaa
     3781 aggaaatggc cagcagcggc ctaccaagca gaaatccgaa gaacagctgc aggcttacta
     3841 caagaagacc ctggaagaga gactgagcgc cggagccctg aacacctttc ctagagcctt
     3901 caaacaggtg atggaaggcc gggacataaa gctggtcatc aaccagacag cccaggattg
     3961 cttcggctgt ctggagagca tctcccagat taggaccgct acacgggata agaaggatac
     4021 cgtgacaaga gagaaacacc ctaagaagcc cttccagaag tggatgaaag acagagcaat
     4081 caagaaaggc aattacctga gattccagag actgttttac ctggacagag gcaagctggc
     4141 caaaatcatt ctggacgaca tcgagtgtct gagttgcgac atccctctgt ccgagatcta
     4201 cagcgtgttc aaaactcggt gggaaacaac aggcagcttc aagtctctgg gcgacttcaa
     4261 gacatacggc aaagccgata acacggcttt tagagagctg atcaccgcca aggagattga
     4321 aaagaacgtg caggagatga gcaagggcag cgcccctggc cccgatggca tcaccctggg
     4381 agatgtggtg aagatggacc ctgagttcag cagaacgatg gagattttca atctgtggct
     4441 gaccaccggc aagattcccg acatggtgcg gggatgtcgg accgtgctga tcccaaagag
     4501 ctccaagccc gatagactga aggacatcaa caactggaga cccatcacca tcggcagcat
     4561 cctactcaga ctgtttagca gaatcgtgac cgccagactg tccaaggctt gtcctctgaa
     4621 tcctaggcag agaggcttca tccgggctgc tggttgcagc gagaacctga agctgttgca
     4681 gaccatcatc tggagcgcca agagagagca cagacctctg ggcgtggtgt ttgtggacat
     4741 cgccaaagcc ttcgacaccg tgtcccacca gcatatcatc cacgtgctgc aacaaagaga
     4801 ggtggacccc cacatcgtgg gcctggtcag caacatgtac gagaacatca gcacctacat
     4861 cacaaccaag cggaacaccc acaccgacaa gatccagatc cgggtgggcg tgaagcaggg
     4921 cgatcctatg tcccctcttc tgttcaacct ggccatggac cccctgttgt gcaagctgga
     4981 ggagagcggc aagggctacc accggggcca gagcagcatc acagccatgg cgttcgccga
     5041 cgatttggtg ttgctgagcg attcttggga gaacatgaac accaacatca gcatcctgga
     5101 gaccttctgc aacctgaccg gcctgaaaac ccagggccag aagtgccacg gcttctatat
     5161 caagcctaca aaagacagct acaccatcaa tgactgcgcc gcctggacca tcaacggcac
     5221 acctctgaat atgattgacc ctggcgaaag cgagaagtac ctgggcttga gattcgaccc
     5281 ttggatcgga atcgccagaa gcggcctgag caccaagctc gacttctggt tgcagagaat
     5341 cgaccaggca ccactgaagc ccctgcaaaa gacagacatc ctgaagacat acaccatccc
     5401 tcgcctaatc tatatcgccg accactctga ggtgaaaacc gccctgctgg aaaccctgga
     5461 ccaaaagatc cggacagccg tgaaggaatg gctgcacctg ccgccttgta catgcgacgc
     5521 tatcctgtac agcagcacca gagatggcgg gctgggcatc actaagctgg ccggcctcat
     5581 tcctagcgtg caggccagac ggctgcatag aatcgcacaa agctctgatg acaccatgaa
     5641 gtgctttatg gagaaagaga aaatggaaca gctgcacaag aagctgtgga ttcaggccag
     5701 aggagaccga gaaaacatcc catctatctg ggaggctcct cctagctctg agcctcctaa
     5761 caacgtgtct acaaacagcg agtgggaggc ccctacccag aaggacaagt tccccaagcc
     5821 atgtaactgg cggaaaaacg agttcaagaa gtggaccaag ctggccagcc agggccgggg
     5881 cattgtgaac ttcgagcggg acaagatctc taaccactgg attcagtact atagaagaat
     5941 ccctcaccgg aagctgctga ccgccctgca gctgagagct aatgtatacc ccacaagaga
     6001 gttcctggcc agaggaagac aggaccagta catcaaagcc tgcagacact gcgatgctga
     6061 catcgaaagc tgtgcacata tcatcggcaa ctgccccgtg acccaggacg cccgcataaa
     6121 gcgtcacaac tatatctgcg agcttctgct ggaagaagcc aaaaagaaag attgggtggt
     6181 gtttaaggaa cctcacatcc gggacagcaa caaggagctg tacaagcctg atctgatctt
     6241 cgtcaaggac gccagagctc tggttgtgga cgtgactgtg agatacgagg ccgctaagtc
     6301 ctccctggaa gaggccgccg ccgagaaggt gagaaagtac aaacacctgg aaaccgaggt
     6361 gagacacctg accaacgcca aagacgtgac cttcgtcggt ttccctctgg gcgctcgcgg
     6421 caagtggcac caggacaact tcaagctcct gactgaactg ggactgtcta aaagcagaca
     6481 ggtgaagatg gcagagacat tttccaccgt ggctctgttc tcctccgtgg acatcgtcca
     6541 catgttcgcc agcagagccc gaaaaagcat ggtgatggct agcccaaaga agaagaggaa
     6601 agtcggatcc gactacaaag accatgacgg tgattataaa gatcatgaca tcgattacaa
     6661 ggatgacgat gacaagtaat ctagactgca gcgcggggat ctcatgctgg agttcttcgc
     6721 ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     6781 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     6841 tgtatcttat taattaacgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     6901 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     6961 aaagtgtaaa gcctagggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     7021 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     7081 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     7141 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     7201 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     7261 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     7321 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     7381 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     7441 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     7501 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     7561 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     7621 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     7681 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     7741 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     7801 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     7861 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     7921 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     7981 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     8041 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     8101 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     8161 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     8221 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     8281 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     8341 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     8401 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     8461 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     8521 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     8581 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     8641 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     8701 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     8761 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     8821 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     8881 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     8941 catttatcag ggttattgtc tcatgagcgg atacatattt gaatatattt agaaaaataa
     9001 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg
     9061 agatcgatct cccgatcccc tagggtcgac tctcagtaca atctgctctg atgccgcata
     9121 gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa
     9181 aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt
     9241 taggcgtttt gcgctgcttc gcgatgtacg ggccagatat
//
