LOCUS       sequence-472649-        5879 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  300bp donor RNA complementary to Cas9 cut site at AAVS1 (Addgene 
            plasmid #72833) with intronic sequence intervening the 3bp insertion
            signature (ATC). Can be used to measure RT-DSBR in human cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5879)
  AUTHORS   Jalan M, Brambati A, Shah H, McDermott N, Patel J, Zhu Y, Doymaz A, 
            Wu J, Anderson KS, Gazzo A, Pareja F, Yamaguchi TN, Vougiouklakis T,
            Ahmed-Seghir S, Steinberg P, Neiman-Golden A, Azeroglu B, 
            Gomez-Aguilar J, da Silva EM, Hussain S, Higginson D, Boutros PC, 
            Riaz N, Reis-Filho JS, Powell SN, Sfeir A
  TITLE     RNA transcripts serve as a template for double-strand break repair 
            in human cells.
  JOURNAL   Nat Commun. 2025 May 10;16(1):4349. doi: 10.1038/s41467-025-59510-x.
  PUBMED    40348775
REFERENCE   2  (bases 1 to 5879)
  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: "Nat 
            Commun."; date: "2025-05-10"; volume: "16(1):4349. doi"; pages: " 
            10.1038/s41467-025-59510-x"
COMMENT     Sequence Label: pMJ1.19 (AAVS1 RNA donor in pCDNA3.1)
FEATURES             Location/Qualifiers
     source          1..5879
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1470..1487)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1476..1700
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      1746..2174
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1833..1852)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2043..2064
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2183..2203)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2188..2517
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2368..2503
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2430..2449
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2584..3378
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2638..2657)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3248..3267
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    3552..3673
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3589..3608)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3643..3662
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3722..3738)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3722..3738)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3735..3757)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3746..3762
                     /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(3770..3776,3777..3794,3795..3800))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3815..3836
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3953..3970)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4124..4712)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4204..4223)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4883..5674,5675..5743))
                     /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     5506..5525
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5744..5848)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc
      901 gtttaaactt aagcttggta ccgagctcgg atccactagt ccagtgtggt ggaattctgc
      961 agatatctta gaggttctgg caaggagaga gatggctcca ggaaatgggg gtgtgtcacc
     1021 agataaggaa tctgcctaac aggaggtggg ggttagaccc aatatcagga gactaggaag
     1081 gaggaggcct aaggatgggg cttttctgtc accaatcggt aagtgttggt atcaaggtta
     1141 caagacaggt ttaaggagac caatagaaac tgggcatgtg gagacagaga agactcttgg
     1201 gtttctgata ggcactgact ctctctgcct attggtctgt ctttttctgt gtggcagatc
     1261 tgtccctagt ggccccactg tggggtggag gggacagata aaagtaccca gaaccagagc
     1321 cacattaacc ggccctggga atataaggtg gtcccagctc ggggacacag gatccctgga
     1381 ggcagcaaac atgctgtcct gaagtggacg atatccagca cagtggcggc cgctcgagtc
     1441 tagagggccc gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc
     1501 tgttgtttgc ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct
     1561 ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg
     1621 gggtggggtg gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg
     1681 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     1741 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     1801 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     1861 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     1921 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag
     1981 tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa
     2041 tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga
     2101 tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa
     2161 atttaacgcg aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc
     2221 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga
     2281 aagtccccag gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
     2341 accatagtcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     2401 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc
     2461 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag
     2521 ctcccgggag cttgtatatc cattttcgga tctgatcaag agacaggatg aggatcgttt
     2581 cgcatgattg aacaagatgg attgcacgca ggttctccgg ccgcttgggt ggagaggcta
     2641 ttcggctatg actgggcaca acagacaatc ggctgctctg atgccgccgt gttccggctg
     2701 tcagcgcagg ggcgcccggt tctttttgtc aagaccgacc tgtccggtgc cctgaatgaa
     2761 ctgcaggacg aggcagcgcg gctatcgtgg ctggccacga cgggcgttcc ttgcgcagct
     2821 gtgctcgacg ttgtcactga agcgggaagg gactggctgc tattgggcga agtgccgggg
     2881 caggatctcc tgtcatctca ccttgctcct gccgagaaag tatccatcat ggctgatgca
     2941 atgcggcggc tgcatacgct tgatccggct acctgcccat tcgaccacca agcgaaacat
     3001 cgcatcgagc gagcacgtac tcggatggaa gccggtcttg tcgatcagga tgatctggac
     3061 gaagagcatc aggggctcgc gccagccgaa ctgttcgcca ggctcaaggc gcgcatgccc
     3121 gacggcgagg atctcgtcgt gacccatggc gatgcctgct tgccgaatat catggtggaa
     3181 aatggccgct tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag
     3241 gacatagcgt tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc
     3301 ttcctcgtgc tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt
     3361 cttgacgagt tcttctgagc gggactctgg ggttcgaaat gaccgaccaa gcgacgccca
     3421 acctgccatc acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa
     3481 tcgttttccg ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct
     3541 tcgcccaccc caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca
     3601 caaatttcac aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca
     3661 tcaatgtatc ttatcatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat
     3721 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     3781 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     3841 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     3901 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     3961 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     4021 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     4081 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4141 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4201 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4261 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4321 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4381 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4441 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4501 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4561 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4621 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4681 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4741 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4801 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4861 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4921 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4981 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     5041 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     5101 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5161 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5221 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5281 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5341 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5401 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5461 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5521 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5581 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5641 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5701 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5761 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5821 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtc
//
