LOCUS       sequence-442276-        4773 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In vitro transcription of the optimized R2Tg RNA donor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4773)
  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 4773)
  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-donor_opt(R2Tg)-GFP
FEATURES             Location/Qualifiers
     source          1..4773
                     /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"
     polyA_signal    741..789
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     complement(907..926)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(join(953..1666,1667..1669,1670..1672))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             complement(join(956..1666,1667..1669,1670..1672))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(998..1019)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1347..1366
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1606..1627
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1669..1678
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1745..1948)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(1774..1794)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        1949..2252
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     complement(2552..2568)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2552..2568)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2565..2587)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2576..2592
                     /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(2600..2606,2607..2624,2625..2630))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2645..2666
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2783..2800)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2954..3542)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3034..3053)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3713..4504,4505..4573))
                     /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     4336..4355
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4574..4678)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4746..4764
                     /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 ttgacgcgat gtgatttctg
      481 cccagtgctc tgaatgtcaa agtgaagaaa ttcaatgaag cgcgggtaaa cggcgggagt
      541 aactatgact ctcttaaggg tctagttaca actgggcatc gctgcagaga tcgcacctcc
      601 tcgtggtccc gctggtagcc cttcgaaggg tgactaagtc gatctctgcc ccaggtacgg
      661 agccgttggg actcaccagt ccaacgtaac tcctgcctaa attcggtgaa acaaattcct
      721 cggtaaaaag ccccggtacc gaacaaacga cccaacaccc gtgcgtttta ttctgtcttt
      781 ttattgccga tcccctcaga agaactcgtc aagaaggcga tagaaggcga tgcgctgcga
      841 atcgggagcg gcgataccgt aaagcacgag gaagcggtca gcccattcgc cgccaagctc
      901 ttcagcaata tcacgggtag ccaacgctat gtcctgatag cggtcggccg ctttacttgt
      961 acagctcgtc catgccgaga gtgatcccgg cggcggtcac gaactccagc aggaccatgt
     1021 gatcgcgctt ctcgttgggg tctttgctca gggcggactg ggtgctcagg tagtggttgt
     1081 cgggcagcag cacggggccg tcgccgatgg gggtgttctg ctggtagtgg tcggccagct
     1141 gcacgctgcc gtcctcgatg ttgtggcgga tcttgaagtt caccttgatg ccgttcttct
     1201 gcttgtcggc catgatatag acgttgtggc tgttgtagtt gtactccagc ttgtgcccca
     1261 ggatgttgcc gtcctccttg aagtcgatgc ccttcagctc gatgcggttc accagggtgt
     1321 cgccctcgaa cttcacctcg gcgcgggtct tgtagttgcc gtcgtccttg aagaagatgg
     1381 tgcgctcctg gacgtagcct tcgggcatgg cggacttgaa gaagtcgtgc tgcttcatgt
     1441 ggtcggggta gcggctgaag cactgcacgc cgtaggtcag ggtggtcacg agggtgggcc
     1501 agggcacggg cagcttgccg gtggtgcaga tgaacttcag ggtcagcttg ccgtaggtgg
     1561 catcgccctc gccctcgccg gacacgctga acttgtggcc gtttacgtcg ccgtccagct
     1621 cgaccaggat gggcaccacc ccggtgaaca gctcctcgcc cttgctcacc atggtggcga
     1681 attcgaagct tgagctcgag atctgagtcc ggtagcgcta gcggatctga cggttcacta
     1741 aaccagctct gcttatatag acctcccacc gtacacgcct accgcccatt tgcgtcaatg
     1801 gggcggagtt gttacgacat tttggaaagt cccgttgatt ttggtgccaa aacaaactcc
     1861 cattgacgtc aatggggtgg agacttggaa atccccgtga gtcaaaccgc tatccacgcc
     1921 cattgatgta ctgccaaaac cgcatcacca tggtaatagc gatgactaat acgtagatgt
     1981 actgccaagt aggaaagtcc cataaggtca tgtactgggc ataatgccag gcgggccatt
     2041 taccgtcatt gacgtcaata gggggcgtac ttggcatatg atacacttga tgtactgcca
     2101 agtgggcagt ttaccgtaaa tactccaccc attgacgtca atggaaagtc cctattggcg
     2161 ttactatggg aacatacgtc attattgacg tcaatgggcg ggggtcgttg ggcggtcagc
     2221 caggcgggcc atttaccgta agttatgtaa cgcccgggca cgggatttgt ccaaggtgga
     2281 cgggccacct ttacttaacc cggaaaagga acatatataa tttatgtgtg ttcgataaat
     2341 agccaaatgc ctcgtcatct aattagtgac gcgcatgaat ggatgaacga gattcccact
     2401 gtccctacct actatccagc gaaaccacag ccaagggaaa gctgccttct gcggggcttg
     2461 ccttctggcc atgcccttct tctctccctt gcacctgtac ctcttggtct ttgaataaag
     2521 cctgagtagg aagaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     2581 atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg
     2641 cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg
     2701 gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc
     2761 gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc
     2821 ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata
     2881 acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg
     2941 cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct
     3001 caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa
     3061 gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc
     3121 tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt
     3181 aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg
     3241 ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg
     3301 cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct
     3361 tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc
     3421 tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg
     3481 ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     3541 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     3601 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     3661 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     3721 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     3781 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     3841 caatgatacc gcgagagcca cgctcaccgg ctccagattt atcagcaata aaccagccag
     3901 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     3961 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     4021 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     4081 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     4141 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     4201 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     4261 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     4321 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     4381 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     4441 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     4501 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     4561 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     4621 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     4681 catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct
     4741 ataaaaatag gcgtatcacg aggccctttc gtc
//
