LOCUS       sequence-374347-        6044 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of nuclear ZF-DdCBE in mammalian cells (N-terminal DddAN,
            Left).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6044)
  AUTHORS   Willis JCW, Silva-Pinheiro P, Widdup L, Minczuk M, Liu DR
  TITLE     Compact zinc finger base editors that edit mitochondrial or nuclear 
            DNA in vitro and in vivo.
  JOURNAL   Nat Commun. 2022 Nov 23;13(1):7204. doi: 10.1038/s41467-022-34784-7.
  PUBMED    36418298
REFERENCE   2  (bases 1 to 6044)
  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: "2022-11-23"; volume: "13(1):7204. doi"; pages: " 
            10.1038/s41467-022-34784-7"
COMMENT     Sequence Label: pL5N_LT32-HBB_X1
FEATURES             Location/Qualifiers
     source          1..6044
                     /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"
     CDS             940..960
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             970..996
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             1039..1059
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1069..1095
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     CDS             1774..2025
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     rep_origin      2276..2704
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2363..2382)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2573..2594
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2713..2733)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2718..3047
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2898..3033
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2960..2979
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        3095..3142
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             3161..3559
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3717..3838
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3754..3773)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3808..3827
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3887..3903)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3887..3903)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3900..3922)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3911..3927
                     /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(3935..3941,3942..3959,3960..3965))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3980..4001
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4118..4135)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4289..4877)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4369..4388)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5048..5839,5840..5908))
                     /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     5671..5690
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5909..6013)
                     /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 accatgaaac ggacagccga cggaagcgag ttcgagtcac caaagaagaa gcggaaagtc
      961 agcggatctc ctgcagctaa gagagtgaag ctggactctg gttcaaagag aactgctgat
     1021 ggttctgaat ttgaatctcc taaaaaaaaa agaaaggttt caggcagccc agctgcaaaa
     1081 agggttaaat tggatagtgg gtccgccgct tctggcggct caggcagcta cgccctgggt
     1141 ccgtatcaga ttagcgcccc gcagctgcca gcttacaatg gtcagaccgt gggtaccttc
     1201 tactatgtga acgacgcggg cggtctggag agcaaggtgt ttagcagcgg cggtccaacc
     1261 ccgtacccaa actatgccaa tgccggtcat gtggagggtc agagcgccct gttcatgcgt
     1321 gataacggca tcagcgaggg tctggtgttc cacaacaacc cggaaggcac ctgcggtttt
     1381 tgcgtgaaca tgatcgagac cctgctgccg gaaaacgcga aaatgaccgt ggtgccgccg
     1441 aaaggtggat ccggcggagg aggttctggc ggttctggag gaagtatggc tgagaggccc
     1501 ttcgcctgcg atatatgtgg caggaaattc gctcaaagcg gggacttgag aaggcacatc
     1561 cgcacccaca ctggcgaaaa gccgtttgcg tgtgacatct gcggacgcaa gtttgcgcgg
     1621 agcgaccatc tcactactca tataaggaca cacaccggtg agaaaccctt cgcttgcgac
     1681 atttgcggga gaaagtttgc agatcccgga cacttggttc gacataccaa gatacacctg
     1741 cggagcggag gatccggagg atctggaggc agcactaatc tgagcgacat cattgagaag
     1801 gagactggga aacagctggt cattcaggag tccatcctga tgctgcctga ggaggtggag
     1861 gaagtgatcg gcaacaagcc agagtctgac atcctggtgc acaccgccta cgacgagtcc
     1921 acagatgaga atgtgatgct gctgacctct gacgcccccg agtataagcc ttgggccctg
     1981 gtcatccagg attctaacgg cgagaataag atcaagatgc tgtgataaac cacgatcgtt
     2041 atgctgatca taccctaatg atcccagcaa gataatgtcc tgtcttctaa gatgtgcatc
     2101 aagcctggta catactgaaa accctataag gtcctggata atttttgttt gattattcat
     2161 tgaagaaaca tttattttcc aattgtgtga agtttttgac tgttaataaa agaatctgtc
     2221 aaccatcaaa cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     2281 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtca cgcgcagcgt gaccgctaca
     2341 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2401 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2461 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg
     2521 ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc
     2581 ttgttccaaa ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg
     2641 attttgccga tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg
     2701 aattaattct gtggaatgtg tgtcagttag ggtgtggaaa gtccccaggc tccccagcag
     2761 gcagaagtat gcaaagcatg catctcaatt agtcagcaac caggtgtgga aagtccccag
     2821 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accatagtcc
     2881 cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat tctccgcccc
     2941 atggctgact aatttttttt atttatgcag aggccgaggc cgcctctgcc tctgagctat
     3001 tccagaagta gtgaggaggc ttttttggag gcctaggctt ttgcaaaaag ctcccgggag
     3061 cttgtatatc cattttcgga tctgatcagc acgtgttgac aattaatcat cggcatagta
     3121 tatcggcata gtataatacg gcaaggtgag gaactaaacc atggccaagc ctttgtctca
     3181 agaagaatcc accctcattg aaagagcaac ggctacaatc aacagcatcc ccatctctga
     3241 agactacagc gtcgccagcg cagctctctc tagcgacggc cgcatcttca ctggtgtcaa
     3301 tgtatatcat tttactgggg gaccttgtgc agaactcgtg gtgctgggca ctgctgctgc
     3361 tgcggcagct ggcaacctga cttgtatcgt cgcgatcgga aatgagaaca ggggcatctt
     3421 gagcccctgc ggacggtgcc gacaggtgct tctcgatctg catcctggga tcaaagccat
     3481 agtgaaggac agtgatggac agccgacggc agttgggatt cgtgaattgc tgccctctgg
     3541 ttatgtgtgg gagggctaag cacttcgtgg ccgaggagca ggactgacac gtgctacgag
     3601 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg
     3661 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcc caccccaact
     3721 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3781 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     3841 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     3901 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3961 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     4021 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     4081 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     4141 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     4201 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     4261 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     4321 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     4381 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     4441 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     4501 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     4561 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     4621 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4681 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4741 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4801 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4861 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4921 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4981 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     5041 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     5101 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     5161 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     5221 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     5281 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     5341 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     5401 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     5461 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     5521 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     5581 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     5641 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5701 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5761 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5821 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5881 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5941 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     6001 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtc
//
