LOCUS       sequence-454054-        6298 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  This plasmid can be used for control experiments and can also be 
            used as a plasmid backbone.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6298)
  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 6298)
  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: mCherry-pCAG-mock
FEATURES             Location/Qualifiers
     source          1..6298
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1..225
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(232..942)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(510..529)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     779..797
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     CDS             complement(943..996)
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             complement(997..1593)
                     /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(1077..1097)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     1574..1593
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(1602..1931)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1616..1751
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        1939..2318
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        2320..2595
                     /label=chicken beta-actin promoter
     intron          2596..3612
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     3536..3558
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     3620..3639
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     promoter        3673..3691
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     polyA_signal    3746..3867
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3783..3802)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3837..3856
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3910..3926)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3910..3926)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3923..3945)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3934..3950
                     /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(3958..3964,3965..3982,3983..3988))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4003..4024
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4141..4158)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4312..4900)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4392..4411)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5071..5862,5863..5931))
                     /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     5694..5713
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5932..6036)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6115..6134)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ccatagagcc caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc
       61 tgtcctgccc caccccaccc cccagaatag aatgacacct actcagacaa tgcgatgcaa
      121 tttcctcatt ttattaggaa aggacagtgg gagtggcacc ttccagggtc aaggaaggca
      181 cgggggaggg gcaaacaaca gatggctggc aactagaagg cacagggatc cttacttgta
      241 cagctcgtcc atgccgccgg tggagtggcg gccctcggcg cgttcgtact gttccacgat
      301 ggtgtagtcc tcgttgtggg aggtgatgtc caacttgatg ttgacgttgt aggcgccggg
      361 cagctgcacg ggcttcttgg ccttgtaggt ggtcttgacc tcagcgtcgt agtggccgcc
      421 gtccttcagc ttcagcctct gcttgatctc gcccttcagg gcgccgtcct cggggtacat
      481 ccgctcggag gaggcctccc agcccatggt cttcttctgc attacggggc cgtcggaggg
      541 gaagttggtg ccgcgcagct tcaccttgta gatgaactcg ccgtcctgca gggaggagtc
      601 ctgggtcacg gtcaccacgc cgccgtcctc gaagttcatc acgcgctccc acttgaagcc
      661 ctcggggaag gacagcttca agtagtcggg gatgtcggcg gggtgcttca cgtaggcctt
      721 ggagccgtac atgaactgag gggacaggat gtcccaggcg aagggcaggg ggccaccctt
      781 ggtcaccttc agcttggcgg tctgggtgcc ctcgtagggg cggccctcgc cctcgccctc
      841 gatctcgaac tcgtggccgt tcacggagcc ctccatgtgc accttgaagc gcatgaactc
      901 cttgatgatg gccatgttat cctcctcgcc cttgctcacc attgggccgg gattctcctc
      961 cacgtcaccg catgttagaa gacttcctct gccctcggca ccgggcttgc gggtcatgca
     1021 ccaggtgcgc ggtccttcgg gcacctcgac gtcggcggtg acggtgaagc cgagccgctc
     1081 gtagaagggg aggttgcggg gcgcggaggt ctccaggaag gcgggcaccc cggcgcgctc
     1141 ggccgcctcc actccgggga gcacgacggc gctgcccaga cccttgccct ggtggtcggg
     1201 cgagacgccg acggtggcca ggaaccacgc gggctccttg ggccggtgcg gcgccaggag
     1261 gccttccatc tgttgctgcg cggccagccg ggaaccgctc aactcggcca tgcgcgggcc
     1321 gatctcggcg aacaccgccc ccgcttcgac gctctccggc gtggtccaga ccgccaccgc
     1381 ggcgccgtcg tccgcgaccc acaccttgcc gatgtcgagc ccgacgcgcg tgaggaagag
     1441 ttcttgcagc tcggtgaccc gctcgatgtg gcggtccgga tcgacggtgt ggcgcgtggc
     1501 ggggtagtcg gcgaacgcgg cggcgagggt gcgtacggcc cgggggacgt cgtcgcgggt
     1561 ggcgaggcgc accgtgggct tgtactcggt catggtcgcg ttttgcaaaa gcctaggcct
     1621 ccaaaaaagc ctcctcacta cttctggaat agctcagagg ccgaggcggc ctcggcctct
     1681 gcataaataa aaaaaattag tcagccatgg ggcggagaat gggcggaact gggcggagtt
     1741 aggggcggga tgggcggagt taggggcggg actatggttg ctgactaatt gagatgcatg
     1801 ctttgcatac ttctgcctgc tggggagcct ggggactttc cacacctggt tgctgactaa
     1861 ttgagatgca tgctttgcat acttctgcct gctggggagc ctggggactt tccacaccct
     1921 aactgacaca cacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
     1981 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
     2041 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
     2101 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
     2161 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
     2221 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
     2281 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt cgaggtgagc cccacgttct
     2341 gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt
     2401 aattattttg tgcagcgatg ggggcggggg gggggggggs gcgcgccagg cggggcgggg
     2461 cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc
     2521 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga
     2581 agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc
     2641 gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg
     2701 gacggccctt ctcctccggg ctgtaattag cgcttggttt aatgacggct tgtttctttt
     2761 ctgtggctgc gtgaaagcct tgaggggctc cgggagggcc ctttgtgcgg ggggagcggc
     2821 tcggggggtg cgtgcgtgtg tgtgtgcgtg gggagcgccg cgtgcggctc cgcgctgccc
     2881 ggcggctgtg agcgctgcgg gcgcggcgcg gggctttgtg cgctccgcag tgtgcgcgag
     2941 gggagcgcgg ccgggggcgg tgccccgcgg tgcggggggg gctgcgaggg gaacaaaggc
     3001 tgcgtgcggg gtgtgtgcgt gggggggtga gcagggggtg tgggcgcgtc ggtcgggctg
     3061 caaccccccc tgcacccccc tccccgagtt gctgagcacg gcccggcttc gggtgcgggg
     3121 ctccgtacgg ggcgtggcgc ggggctcgcc gtgccgggcg gggggtggcg gcaggtgggg
     3181 gtgccgggcg gggcggggcc gcctcgggcc ggggagggct cgggggaggg gcgcggcggc
     3241 ccccggagcg ccggcggctg tcgaggcgcg gcgagccgca gccattgcct tttatggtaa
     3301 tcgtgcgaga gggcgcaggg acttcctttg tcccaaatct gtgcggagcc gaaatctggg
     3361 aggcgccgcc gcaccccctc tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag
     3421 gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag
     3481 cctcggggct gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc
     3541 ggcttctggc gtgtgaccgg cggctctagc gcctctgcta accatgttca tgccttcttc
     3601 tttttcctac agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa
     3661 gctagtgaat tctaatacga ctcactatag gaccggtgct agcctgcagc gcggggatct
     3721 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     3781 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     3841 tttgtccaaa ctcatcaatg tatcttatta attaacgtcg acctctagct agagcttggc
     3901 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     3961 catacgagcc ggaagcataa agtgtaaagc ctagggtgcc taatgagtga gctaactcac
     4021 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4081 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     4141 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4201 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4261 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     4321 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     4381 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     4441 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     4501 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     4561 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     4621 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     4681 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     4741 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     4801 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     4861 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     4921 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     4981 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5041 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5101 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5161 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     5221 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     5281 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     5341 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     5401 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     5461 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     5521 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     5581 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     5641 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     5701 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     5761 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     5821 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     5881 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     5941 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6001 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6061 tgacgtcgac ggatcgggag atcgatctcc cgatccccta gggtcgactc tcagtacaat
     6121 ctgctctgat gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc
     6181 tgagtagtgc gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat
     6241 gaagaatctg cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatat
//
