LOCUS       sequence-381542-        7359 bp    DNA     circular SYN 15-APR-2025
DEFINITION  CMV-TadA 8e-nNcCas9. A-to-G base editing in human cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7359)
  AUTHORS   Liu Z, Chen S, Xie W, Yu H, Lai L, Li Z
  TITLE     Versatile and efficient genome editing with Neisseria cinerea Cas9.
  JOURNAL   Commun Biol. 2022 Nov 26;5(1):1296. doi: 10.1038/s42003-022-04258-z.
  PUBMED    36435853
REFERENCE   2  (bases 1 to 7359)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Commun 
            Biol."; date: "2022-11-26"; volume: "5(1):1296. doi"; pages: " 
            10.1038/s42003-022-04258-z"
COMMENT     Sequence Label: nNc-ABE
FEATURES             Location/Qualifiers
     source          1..7359
                     /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             4345..4365
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4374..4391
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4414..4431)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4420..4644
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4715..4731)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4715..4731)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4728..4750)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4739..4755
                     /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(4763..4769,4770..4787,4788..4793))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4808..4829
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4946..4963)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5117..5705)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5197..5216)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5876..6667,6668..6736))
                     /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     6499..6518
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6737..6841)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6920..6939)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7111..7359,1..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 aagtctctga ggtggagttt
      481 tcccacgagt actggatgag acatgccctg accctggcca agagggcacg ggatgagagg
      541 gaggtgcctg tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggaac
      601 agagccatcg gcctgcacga cccaacagcc catgccgaaa ttatggccct gagacagggc
      661 ggcctggtca tgcagaacta cagactgatt gacgccaccc tgtacgtgac attcgagcct
      721 tgcgtgatgt gcgccggcgc catgatccac tctaggatcg gccgcgtggt gtttggcgtg
      781 aggaactcaa aaagaggcgc cgcaggctcc ctgatgaacg tgctgaacta ccccggcatg
      841 aatcaccgcg tcgaaattac cgagggaatc ctggcagatg aatgtgccgc cctgctgtgc
      901 gatttctatc ggatgcctag acaggtgttc aatgctcaga agaaggccca gagctccatc
      961 aactccggag gatctagcgg aggctcctct ggctctgaga cacctggcac aagcgagagc
     1021 gcaacacctg aaagcagcgg gggcagcagc ggggggtcag ccgccttcaa accaaaccct
     1081 atgaactaca tcctggggct ggccatcgga atcgcaagcg tgggctgggc tatcgtggaa
     1141 atcgacgagg aggagaaccc aatccggctg atcgacctgg gcgtgagagt gtttgagagg
     1201 gcagaggtgc caaagaccgg cgattccctg gcagcagcac ggagactggc acggtctgtg
     1261 aggcgcctga cacggagaag ggcacacaga ctgctgaggg cacgccggct gctgaagaga
     1321 gagggcgtgc tgcaggcagc agacttcgat gagaacggcc tgatcaagag cctgcctaat
     1381 accccatggc agctgagagc agccgccctg gacaggaagc tgacaccact ggagtggtcc
     1441 gccgtgctgc tgcacctgat caagcaccgc ggctacctgt ctcagcggaa gaatgagggc
     1501 gagacagccg ataaggagct gggcgccctg ctgaagggag tggccgacaa cacacacgca
     1561 ctgcagaccg gcgacttccg gacacctgca gagctggccc tgaataagtt cgagaaggag
     1621 agcggacaca tcaggaacca gaggggcgac tattcccaca ccttcaaccg caaggatctg
     1681 caggccgagc tgaatctgct gtttgagaag cagaaggagt tcggcaatcc tcacgtgtcc
     1741 gacggcctga aggagggcat cgagacactg ctgatgaccc agaggccagc cctgtctggc
     1801 gatgcagtgc agaagatgct gggccactgc acatttgagc ccaccgagcc taaggccgcc
     1861 aagaacacct acacagccga gaggttcgtg tggctgacaa agctgaacaa tctgaggatc
     1921 ctggagcagg gaagcgagag gcctctgacc gacacagaga gggccaccct gatggatgag
     1981 ccatacagga agtccaagct gacatatgca caggcaagga agctgctgga cctggacgat
     2041 accgccttct ttaagggcct gagatacggc aaggataatg ccgaggcctc tacactgatg
     2101 gagatgaagg cctatcacgc catcagccgc gccctggaga aggagggcct gaaggacaag
     2161 aagtctcctc tgaacctgag cccagagctg caggatgaga tcggcaccgc cttttccctg
     2221 ttcaagaccg acgaggacat cacaggccgc ctgaaggaca gggtgcagcc tgagatcctg
     2281 gaggccctgc tgaagcacat ctcttttgat aagttcgtgc agatcagcct gaaggccctg
     2341 agaaggatcg tgccactgat ggagcagggc aatcgctacg acgaggcctg caccgagatc
     2401 tacggcgatc actatggcaa gaagaacaca gaggagaaga tctatctgcc ccctatccca
     2461 gccgacgaga tcagaaatcc agtggtgctg agggccctga gccaggcacg caaagtgatc
     2521 aacggagtgg tgcgccggta cggctccccc gcaaggatcc acatcgagac agccagagaa
     2581 gtgggcaagt cttttaagga ccggaaggag atcgagaaga gacaggagga gaatagaaag
     2641 gatagggaga agagcgccgc caagttcagg gagtactttc ccaacttcgt gggcgagcct
     2701 aagtccaagg acatcctgaa gctgcgcctg tacgagcagc agcacggcaa gtgtctgtat
     2761 agcggcaagg agatcaacct gggccggctg aatgagaagg gctatgtgga gatcgatcac
     2821 gccctgccct ttagcagaac ctgggacgat tccttcaaca ataaggtgct ggccctgggc
     2881 tctgagaacc agaataaggg caatcagaca ccatacgagt atttcaacgg caaggacaat
     2941 agcagggagt ggcaggagtt taaggcaagg gtggagacaa gcaggttccc acggtctaag
     3001 aagcagcgga tcctgctgca gaagtttgac gaggatggct tcaaggagag aaacctgaat
     3061 gacaccagat acatcaacag gtttctgtgc cagttcgtgg ccgatcacat gctgctgacc
     3121 ggcaagggca agagaagggt gtttgcctcc aacggccaga tcacaaatct gctgaggggc
     3181 ttctggggcc tgagaaaggt gagggcagag aatgacaggc accacgcact ggatgcagtg
     3241 gtggtggcat gtagcaccat cgccatgcag cagaagatca cacgctttgt gcggtataag
     3301 gagatgaacg ccttcgacgg caagaccatc gataaggaga caggcgaggt gctgcaccag
     3361 aaggcccact ttccacagcc ctgggagttc tttgcccagg aagtgatgat ccgggtgttt
     3421 ggcaagccag acggcaagcc cgagttcgag gaggccgata ccccagagaa gctgcggaca
     3481 ctgctggcag agaagctgag ctccagacct gaggccgtgc acaagtacgt gacccctctg
     3541 ttcatctccc gcgccccaaa tcggaagatg tctggccagg gccacatgga gacagtgaag
     3601 tctgccaagc ggctggacga gggcatcagc gtgctgagag tgcccctgac acagctgaag
     3661 ctgaaggatc tggagaagat ggtgaaccgc gagcgggagc ctaagctgta tgaggccctg
     3721 aaggccagac tggaggccca caaggacgat cccgccaagg cctttgccga gcctttctac
     3781 aagtatgaca aggccggcaa tcggacccag caggtgaagg ccgtgagagt ggagcaggtg
     3841 cagaagacag gcgtgtgggt gcacaaccac aatggcatcg ccgacaacgc cacaatcgtg
     3901 cgggtggacg tgttcgagaa gggcggcaag tactatctgg tgccaatcta ctcctggcag
     3961 gtggcaaagg gaatcctgcc agacagagcc gtggtgcagg gcaaggacga ggaggattgg
     4021 accgtgatgg acgattcttt cgagtttaag ttcgtgctgt acgccaacga cctgatcaag
     4081 ctgaccgcca agaagaatga gtttctgggc tatttcgtgt ctctgaacag ggcaacagga
     4141 gcaatcgaca tcaggaccca cgacacagat agcaccaagg gcaagaacgg catctttcag
     4201 agcgtgggcg tgaagaccgc cctgtccttc cagaagtatc agattgacga actgggaaaa
     4261 gagattagac cctgtaggct gaaaaagaga ccacctgtga gatctggcgg ctcaaaaaga
     4321 accgccgacg gcagcgaatt cgagcccaag aagaagagga aagtctaacc ggtcatcatc
     4381 accatcacca ttgagtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     4441 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     4501 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     4561 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     4621 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctcgatac
     4681 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4741 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctagg
     4801 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4861 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4921 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4981 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5041 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5101 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     5161 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     5221 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     5281 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     5341 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5401 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5461 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5521 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5581 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5641 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     5701 ctcaagaaga tcctttgatc ttttctacgg ggtctgacac tcagtggaac gaaaactcac
     5761 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     5821 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     5881 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     5941 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6001 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6061 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6121 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     6181 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     6241 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     6301 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     6361 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     6421 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     6481 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     6541 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     6601 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     6661 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     6721 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     6781 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     6841 gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatcga tctcccgatc
     6901 ccctagggtc gactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     6961 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     7021 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
     7081 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagt tattaatagt
     7141 aatcaattac ggggtcatta gttcatagcc catatatgga gttccgcgtt acataactta
     7201 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
     7261 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
     7321 tacggtaaac tgcccacttg gcagtacatc aagtgtatc
//
