LOCUS       sequence-272344-        6693 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For orthogonal R loop assay (use with Sp-derived base editor, Sp 
            gRNA, and Sa gRNA for assay).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6693)
  AUTHORS   Doman JL, Raguram A, Newby GA, Liu DR
  TITLE     Evaluation and minimization of Cas9-independent off-target DNA 
            editing by cytosine base editors.
  JOURNAL   Nat Biotechnol. 2020 Feb 10. pii: 10.1038/s41587-020-0414-6. doi: 
            10.1038/s41587-020-0414-6.
  PUBMED    32042165
REFERENCE   2  (bases 1 to 6693)
  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 
            Biotechnol. 2020 Feb 10. pii: 10.1038/s41587-020-0414-6. doi: 
            10.1038/s41587-020-0414-6."
COMMENT     Sequence Label: dead Sa Cas9
FEATURES             Location/Qualifiers
     source          1..6693
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             445..465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3679..3699
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3708..3725
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3748..3765)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3754..3978
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4049..4065)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4049..4065)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4062..4084)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4073..4089
                     /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(4097..4103,4104..4121,4122..4127))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4142..4163
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4280..4297)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4451..5039)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4531..4550)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5210..6001,6002..6070))
                     /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     5833..5852
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6071..6175)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6254..6273)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6445..6693,1..131)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca
      421 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtcagcgg ggggtcaggg
      481 aagcgaaatt acattctggg gctggccatt ggcattacat cagtgggcta tggcatcatt
      541 gactacgaga caagggacgt gatcgacgcc ggcgtgagac tgttcaagga ggccaacgtg
      601 gagaacaatg agggccggag atccaagagg ggagcaaggc gcctgaagcg gagaaggcgc
      661 cacagaatcc agagagtgaa gaagctgctg ttcgattaca acctgctgac cgaccactcc
      721 gagctgtctg gcatcaatcc ttatgaggcc agagtgaagg gcctgtccca gaagctgtct
      781 gaggaggagt ttagcgccgc cctgctgcac ctggcaaaga ggagaggcgt gcacaacgtg
      841 aatgaggtgg aggaggacac cggcaacgag ctgtccacaa aggagcagat cagccgcaat
      901 tccaaggccc tggaggagaa gtatgtggcc gagctgcagc tggagcggct gaagaaggat
      961 ggcgaggtga ggggctccat caatcgcttc aagacctctg actacgtgaa ggaggccaag
     1021 cagctgctga aggtgcagaa ggcctaccac cagctggatc agtcctttat cgatacatat
     1081 atcgacctgc tggagacaag gcgcacatac tatgagggac caggagaggg ctctcccttc
     1141 ggctggaagg acatcaagga gtggtacgag atgctgatgg gccactgcac ctattttcca
     1201 gaggagctga gaagcgtgaa gtacgcctat aacgccgatc tgtacaacgc cctgaatgac
     1261 ctgaacaacc tggtcatcac cagggatgag aacgagaagc tggagtacta tgagaagttc
     1321 cagatcatcg agaacgtgtt caagcagaag aagaagccta cactgaagca gatcgccaag
     1381 gagatcctgg tgaacgagga ggacatcaag ggctaccgcg tgacctccac aggcaagcca
     1441 gagttcacca atctgaaggt gtatcacgat atcaaggaca tcacagcccg gaaggagatc
     1501 atcgagaacg ccgagctgct ggatcagatc gccaagatcc tgaccatcta tcagagctcc
     1561 gaggacatcc aggaggagct gaccaacctg aatagcgagc tgacacagga ggagatcgag
     1621 cagatcagca atctgaaggg ctacaccggc acacacaacc tgagcctgaa ggccatcaat
     1681 ctgatcctgg atgagctgtg gcacacaaac gacaatcaga tcgccatctt taaccggctg
     1741 aagctggtgc caaagaaggt ggacctgtcc cagcagaagg agatcccaac cacactggtg
     1801 gacgatttca tcctgtctcc cgtggtgaag cggagcttca tccagagcat caaagtgatc
     1861 aacgccatca tcaagaagta cggcctgccc aatgatatca tcatcgagct ggccagggag
     1921 aagaactcca aggacgccca gaagatgatc aatgagatgc agaagaggaa ccgccagacc
     1981 aatgagcgga tcgaggagat catcagaacc acaggcaagg agaacgccaa gtacctgatc
     2041 gagaagatca agctgcacga tatgcaggag ggcaagtgtc tgtattctct ggaggccatc
     2101 cctctggagg acctgctgaa caatccattc aactacgagg tggatcacat catcccccgg
     2161 agcgtgagct tcgacaattc ttttaacaat aaggtgctgg tgaagcagga ggaggctagc
     2221 aagaagggca ataggacccc tttccagtac ctgtctagct ccgattctaa gatcagctac
     2281 gagacattca agaagcacat cctgaatctg gccaagggca agggccgcat cagcaagacc
     2341 aagaaggagt acctgctgga ggagcgggac atcaacagat tctccgtgca gaaggacttc
     2401 atcaaccgga atctggtgga caccagatac gccacacgcg gcctgatgaa tctgctgcgg
     2461 tcttatttca gagtgaacaa tctggatgtg aaggtgaaga gcatcaacgg cggcttcacc
     2521 tcctttctgc ggagaaagtg gaagtttaag aaggagcgca acaagggcta taagcaccac
     2581 gccgaggatg ccctgatcat cgccaatgcc gacttcatct ttaaggagtg gaagaagctg
     2641 gacaaggcca agaaagtgat ggagaaccag atgttcgagg agaagcaggc cgagagcatg
     2701 cccgagatcg agacagagca ggagtacaag gagattttca tcacacctca ccagatcaag
     2761 cacatcaagg acttcaagga ctacaagtat tctcacaggg tggataagaa gcccaaccgc
     2821 gagctgatca atgacaccct gtatagcaca cggaaggacg ataagggcaa taccctgatc
     2881 gtgaacaatc tgaacggcct gtacgacaag gataatgaca agctgaagaa gctgatcaac
     2941 aagtctcccg agaagctgct gatgtaccac cacgatcctc agacatatca gaagctgaag
     3001 ctgatcatgg agcagtacgg cgacgagaag aacccactgt ataagtacta tgaggagaca
     3061 ggcaactacc tgacaaagta tagcaagaag gataatggcc ccgtgatcaa gaagatcaag
     3121 tactatggca acaagctgaa tgcccacctg gacatcaccg acgattaccc taactctcgc
     3181 aataaggtgg tgaagctgag cctgaagcca taccggttcg acgtgtacct ggacaacggc
     3241 gtgtataagt ttgtgacagt gaagaatctg gatgtgatca agaaggagaa ctactatgag
     3301 gtgaacagca agtgctacga ggaggccaag aagctgaaga agatcagcaa ccaggccgag
     3361 ttcatcgcct ctttttacaa caatgacctg atcaagatca atggcgagct gtatagagtg
     3421 atcggcgtga acaatgatct gctgaacaga atcgaagtga atatgatcga catcacctac
     3481 agggagtatc tggagaacat gaatgataag aggccccctc gcatcatcaa gaccatcgcc
     3541 tctaagacac agagcatcaa gaagtacagc acagacatcc tggggaacct gtatgaagtc
     3601 aagagcaaga aacatcctca gattatcaag aaaggctctg gcggctcaaa aagaaccgcc
     3661 gacggcagcg aattcgagcc caagaagaag aggaaagtct aaccggtcat catcaccatc
     3721 accattgagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg
     3781 ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt
     3841 cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg
     3901 gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg
     3961 atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctcg ataccgtcga
     4021 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     4081 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tagggtgcct
     4141 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     4201 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     4261 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     4321 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     4381 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4441 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4501 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4561 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4621 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4681 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4741 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4801 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4861 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4921 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4981 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     5041 aagatccttt gatcttttct acggggtctg acactcagtg gaacgaaaac tcacgttaag
     5101 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     5161 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     5221 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     5281 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     5341 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     5401 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5461 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5521 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5581 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5641 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5701 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5761 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5821 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5881 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5941 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     6001 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     6061 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     6121 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     6181 ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tcgatctccc gatcccctag
     6241 ggtcgactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat ctgctccctg
     6301 cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat ttaagctaca acaaggcaag
     6361 gcttgaccga caattgcatg aagaatctgc ttagggttag gcgttttgcg ctgcttcgcg
     6421 atgtacgggc cagatatacg cgttgacatt gattattgac tagttattaa tagtaatcaa
     6481 ttacggggtc attagttcat agcccatata tggagttccg cgttacataa cttacggtaa
     6541 atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata atgacgtatg
     6601 ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag tatttacggt
     6661 aaactgccca cttggcagta catcaagtgt atc
//
