LOCUS       sequence-442278-        7711 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vitro transcription of the optimized R2Tg protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7711)
  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 7711)
  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: IVT-R2Tg_opt
FEATURES             Location/Qualifiers
     source          1..7711
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             469..489
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5281..5301
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5308..5373
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     primer_bind     complement(5490..5506)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5490..5506)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5503..5525)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5514..5530
                     /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(5538..5544,5545..5562,5563..5568))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5583..5604
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5721..5738)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5892..6480)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5972..5991)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6651..7442,7443..7511))
                     /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     7274..7293
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7512..7616)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7684..7702
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt ctaatacgac tcactataag
      421 gaaataagag agaaaagaag agtaagaaga aatataagag ccaccatgcc caagaagaag
      481 aggaaggttc ccgggatgcc taagagaaag gtgtccagcg ccgagggcgc cgccaaagaa
      541 gagcctaagc ggagaagcgc cagactgagc gccaagcctc cagctaaagt ggaagccaag
      601 cccaagaagg ccgctgccaa ggacaagagc tctgataaga aagtccagac caagggaaaa
      661 cggggcgcta aaggcaagca ggccgaggtg gccaaccagg agacaaagga agatctgcct
      721 gctgagaacg gcgaaaccaa gaccgaggaa agccccgcca gcgacgaggc cggcgagaag
      781 gaggccaagt ctgacgccag ctgccccaag ccaggcccac ctgtgtctgc cggcgccatg
      841 agcctggaat ctggactgac aacacacagc gtgctggcca tcgagagagg acccaacagc
      901 ctggccaatt ccggctctga tttcggcggc ggcggcctgg gcctgccact aagactgctg
      961 cgtgtatcag tgggcacgca gaccagcaga agcgattggg tggatctggt gagctggtcc
     1021 caccctggcc ctacaagcaa gtcccagcag gttgaccttg tgagcctgtt cccgaagcac
     1081 cgggtggacc tgctatccaa aaatgatcag gtggatctgg tcgcgcaatt cctgccttca
     1141 aagttccctc ccaacctggc tgaaaatgac ctcgccctgc tggtgaacct ggaattttac
     1201 agaagcgacc tgcacgtgta cgaatgcgtg cacttcgctg cccattggga gggcctgagc
     1261 ggactgccag aagtctacga acagctcgcc cctcagcctt gcgtgggaga gaccctgcat
     1321 agctccctgc ctcgggatag cgagctgttc gtgcctgagg agggctctag cgaaaaggaa
     1381 tcggaagatg cccccaagac cagccccccc acccctggca aacatggcct ggagcagaca
     1441 ggcgaggaga aggtgatggt taccgtcccc gacaagaacc ctccctgtcc ttgctgcggc
     1501 accagagtga acagcgtgct gaatctgatc gagcacctga aggtgagcca cggcaagcgt
     1561 ggagtttgtt tccggtgcgc caagtgcggc aaggaaaaca gcaactacca cagcgtggtg
     1621 tgccactttc ctaagtgtag aggccctgag acagaaaagg cccctgccgg agaatggatt
     1681 tgtgaggtgt gcaaccggga ttttaccacc aagatcggcc tgggccagca caaacggctg
     1741 gcccaccccg ccgtgagaaa ccaggagagg atcgtggcca gccaacctaa agagacaagc
     1801 aacagaggcg cccacaagcg gtgctggacg aaggaagagg aagaattgtt gatcagactg
     1861 gaagcccagt tcgagggaaa caagaacatc aacaagctga ttgccgagca catcaccacc
     1921 aagaccgcca agcagatcag cgacaagaga aggctgttaa gcagaaagcc tgccgaggag
     1981 cccagagagg aacctggcac ctgccaccac acccggagag ccgccgcttc actgcggacc
     2041 gagcccgaga tgtctcacca cgcccaggcc gaggacagag ataacggcgg aggaatgcct
     2101 aagagaaagg tgtccagcgc cgagggcgcc gccaaagaag agcctaagcg gagaagcgcc
     2161 agactgagcg ccaagcctcc agctaaagtg gaagccaagc ccaagaaggc cgctgccaag
     2221 gacaagagct ctgataagaa agtccagacc aagggaaaac ggggcgctaa aggcaagcag
     2281 gccgaggtgg ccaaccagga gacaaaggaa gatctgcctg ctgagaacgg cgaaaccaag
     2341 accgaggaaa gccccgccag cgacgaggcc ggcgagaagg aggccaagtc tgacggagga
     2401 cccggcagac ggcctctccc cggaagggct gccgccggcg gaagaaccat ggatgagatc
     2461 agaagacacc ccgacaaagg aaatggccag cagcggccta ccaagcagaa atccgaagaa
     2521 cagctgcagg cttactacaa gaagaccctg gaagagagac tgagcgccgg agccctgaac
     2581 acctttccta gagccttcaa acaggtgatg gaaggccggg acataaagct ggtcatcaac
     2641 cagacagccc aggattgctt cggctgtctg gagagcatct cccagattag gaccgctaca
     2701 cgggataaga aggataccgt gacaagagag aaacacccta agaagccctt ccagaagtgg
     2761 atgaaagaca gagcaatcaa gaaaggcaat tacctgagat tccagagact gttttacctg
     2821 gacagaggca agctggccaa aatcattctg gacgacatcg agtgtctgag ttgcgacatc
     2881 cctctgtccg agatctacag cgtgttcaaa actcggtggg aaacaacagg cagcttcaag
     2941 tctctgggcg acttcaagac atacggcaaa gccgataaca cggcttttag agagctgatc
     3001 accgccaagg agattgaaaa gaacgtgcag gagatgagca agggcagcgc ccctggcccc
     3061 gatggcatca ccctgggaga tgtggtgaag atggaccctg agttcagcag aacgatggag
     3121 attttcaatc tgtggctgac caccggcaag attcccgaca tggtgcgggg atgtcggacc
     3181 gtgctgatcc caaagagctc caagcccgat agactgaagg acatcaacaa ctggagaccc
     3241 atcaccatcg gcagcatcct actcagactg tttagcagaa tcgtgaccgc cagactgtcc
     3301 aaggcttgtc ctctgaatcc taggcagaga ggcttcatcc gggctgctgg ttgcagcgag
     3361 aacctgaagc tgttgcagac catcatctgg agcgccaaga gagagcacag acctctgggc
     3421 gtggtgtttg tggacatcgc caaagccttc gacaccgtgt cccaccagca tatcatccac
     3481 gtgctgcaac aaagagaggt ggacccccac atcgtgggcc tggtcagcaa catgtacgag
     3541 aacatcagca cctacatcac aaccaagcgg aacacccaca ccgacaagat ccagatccgg
     3601 gtgggcgtga agcagggcga tcctatgtcc cctcttctgt tcaacctggc catggacccc
     3661 ctgttgtgca agctggagga gagcggcaag ggctaccacc ggggccagag cagcatcaca
     3721 gccatggcgt tcgccgacga tttggtgttg ctgagcgatt cttgggagaa catgaacacc
     3781 aacatcagca tcctggagac cttctgcaac ctgaccggcc tgaaaaccca gggccagaag
     3841 tgccacggct tctatatcaa gcctacaaaa gacagctaca ccatcaatga ctgcgccgcc
     3901 tggaccatca acggcacacc tctgaatatg attgaccctg gcgaaagcga gaagtacctg
     3961 ggcttgagat tcgacccttg gatcggaatc gccagaagcg gcctgagcac caagctcgac
     4021 ttctggttgc agagaatcga ccaggcacca ctgaagcccc tgcaaaagac agacatcctg
     4081 aagacataca ccatccctcg cctaatctat atcgccgacc actctgaggt gaaaaccgcc
     4141 ctgctggaaa ccctggacca aaagatccgg acagccgtga aggaatggct gcacctgccg
     4201 ccttgtacat gcgacgctat cctgtacagc agcaccagag atggcgggct gggcatcact
     4261 aagctggccg gcctcattcc tagcgtgcag gccagacggc tgcatagaat cgcacaaagc
     4321 tctgatgaca ccatgaagtg ctttatggag aaagagaaaa tggaacagct gcacaagaag
     4381 ctgtggattc aggccagagg agaccgagaa aacatcccat ctatctggga ggctcctcct
     4441 agctctgagc ctcctaacaa cgtgtctaca aacagcgagt gggaggcccc tacccagaag
     4501 gacaagttcc ccaagccatg taactggcgg aaaaacgagt tcaagaagtg gaccaagctg
     4561 gccagccagg gccggggcat tgtgaacttc gagcgggaca agatctctaa ccactggatt
     4621 cagtactata gaagaatccc tcaccggaag ctgctgaccg ccctgcagct gagagctaat
     4681 gtatacccca caagagagtt cctggccaga ggaagacagg accagtacat caaagcctgc
     4741 agacactgcg atgctgacat cgaaagctgt gcacatatca tcggcaactg ccccgtgacc
     4801 caggacgccc gcataaagcg tcacaactat atctgcgagc ttctgctgga agaagccaaa
     4861 aagaaagatt gggtggtgtt taaggaacct cacatccggg acagcaacaa ggagctgtac
     4921 aagcctgatc tgatcttcgt caaggacgcc agagctctgg ttgtggacgt gactgtgaga
     4981 tacgaggccg ctaagtcctc cctggaagag gccgccgccg agaaggtgag aaagtacaaa
     5041 cacctggaaa ccgaggtgag acacctgacc aacgccaaag acgtgacctt cgtcggtttc
     5101 cctctgggcg ctcgcggcaa gtggcaccag gacaacttca agctcctgac tgaactggga
     5161 ctgtctaaaa gcagacaggt gaagatggca gagacatttt ccaccgtggc tctgttctcc
     5221 tccgtggaca tcgtccacat gttcgccagc agagcccgaa aaagcatggt gatggctagc
     5281 ccaaagaaga agaggaaagt cggatccgac tacaaagacc atgacggtga ttataaagat
     5341 catgacatcg attacaagga tgacgatgac aagtaaaagc tgccttctgc ggggcttgcc
     5401 ttctggccat gcccttcttc tctcccttgc acctgtacct cttggtcttt gaataaagcc
     5461 tgagtaggaa gaagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     5521 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     5581 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     5641 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     5701 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     5761 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     5821 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     5881 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     5941 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     6001 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     6061 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     6121 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     6181 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     6241 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     6301 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     6361 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     6421 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     6481 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     6541 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     6601 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     6661 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     6721 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     6781 atgataccgc gagagccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     6841 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6901 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6961 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     7021 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     7081 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     7141 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     7201 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     7261 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     7321 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     7381 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     7441 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     7501 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     7561 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     7621 tttccccgaa aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat
     7681 aaaaataggc gtatcacgag gccctttcgt c
//
