LOCUS       sequence-461181-        6473 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express HACE Editor as a fusion of AID cytidine deaminase and Ns3h 
            helicase with mTagBFP2 reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6473)
  AUTHORS   Chen XD, Chen Z, Wythes G, Zhang Y, Orr BC, Sun G, Chao YK, Navarro 
            Torres A, Thao K, Vallurupalli M, Sun J, Borji M, Tkacik E, Chen H, 
            Bernstein BE, Chen F
  TITLE     Helicase-assisted continuous editing for programmable mutagenesis of
            endogenous genomes.
  JOURNAL   Science. 2024 Oct 11;386(6718):eadn5876. doi: 
            10.1126/science.adn5876. Epub 2024 Oct 11.
  PUBMED    39388570
REFERENCE   2  (bases 1 to 6473)
  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: "Science."; 
            date: "2024-10-11"; volume: "386(6718):eadn5876. doi"; pages: " 
            10.1126/science.adn5876. Epub 2024 Oct 11"
COMMENT     Sequence Label: pCMV-AID-Ns3h-UGI-T2A-mTagBFP2
FEATURES             Location/Qualifiers
     source          1..6473
                     /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"
     regulatory      384..393
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2433..2681
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             2694..2714
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2715..2768
                     /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             2769..3479
                     /codon_start=1
                     /product="enhanced monomeric blue fluorescent protein 
                     (Subach et al., 2011)"
                     /label=mTagBFP2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTM
                     RIKVVEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGG
                     VLTATQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRND
                     MALKLVGGSHLIANAKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAV
                     ARYCDLPSKLGHKLN"
     CDS             3488..3505
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3528..3545)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3534..3758
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3829..3845)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3829..3845)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3842..3864)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3853..3869
                     /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(3877..3883,3884..3901,3902..3907))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3922..3943
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4060..4077)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4231..4819)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4311..4330)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4990..5781,5782..5850))
                     /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     5613..5632
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5851..5955)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6034..6053)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6225..6473,1..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 ggccgcgaga gccgccacca tggacagcct cttgatgaac cggagggagt
      421 ttctttacca attcaaaaat gtccgctggg ctaagggtcg gcgtgagacc tacctgtgct
      481 acgtagtgaa gaggcgtgac agtgctacat ccttttcact ggactttggt tatcttcgca
      541 ataagaacgg ctgccacgtg gaattgctct tcctccgcta catctcggac tgggacctag
      601 accctggccg ctgctaccgc gtcacctggt tcatctcctg gagcccctgc tacgactgtg
      661 cccgacatgt ggccgacttt ctgcgaggga accccaacct cagtctgagg atcttcaccg
      721 cgcgcctcta cttctgtgag gaccgcaagg ctgagcccga ggggctgcgg cggctgcacc
      781 gcgccggggt gcaaatagcc atcatgacct tcaaagatta tttttactgc tggaatactt
      841 ttgtagaaaa ccacggaaga actttcaaag cctgggaagg gctgcatgaa aattcagttc
      901 gtctctccag acagcttcgg cgcatccttt tgcccctgta tgaggttgat gacttacgag
      961 acgcatttcg tactagcggc agcgagactc ccgggacctc agagtccgcc acacccgaaa
     1021 gtatggtgga cttcataccc gttgagtcta tggaaactac catgcggtct ccggtcttca
     1081 cagacaactc aacccccccg gctgtaccgc agacattcca agtggcacat ctgcacgctc
     1141 ctactggcag cggcaagagc accaaagtgc cggctgcgta tgcagcccaa gggtacaagg
     1201 tgctcgtcct gaacccgtcc gttgccgcca ccttagggtt tggggcgtat atgtccaagg
     1261 cacacggtat cgaccctaac atcagaactg gggtaaggac cattaccacg ggcggctcca
     1321 ttacgtactc cacctatggc aagttccttg ccgacggtgg ctgttctggg ggcgcctatg
     1381 acatcataat atgtgatgag tgccactcaa ctgactcgac taccatcttg ggcatcggca
     1441 cagtcctgga ccaagcggag acggctggag cgcggctcgt cgtgctcgcc accgctacac
     1501 ctccgggatc ggttaccgtg ccacacccca atatcgagga aataggcctg tccaacaatg
     1561 gagagatccc cttctatggc aaagccatcc ccattgaggc catcaagggg gggaggcatc
     1621 tcattttctg ccattccaag aagaaatgtg acgagctcgc cgcaaagctg acaggcctcg
     1681 gactgaacgc tgtagcatat taccggggcc ttgatgtgtc cgtcataccg cctatcggag
     1741 acgtcgttgt cgtggcaaca gacgctctaa tgacgggttt caccggcgat tttgactcag
     1801 tgatcgactg caatacatgt gtcacccaga cagtcgactt cagcttggat cccaccttca
     1861 ccattgagac gacgaccgtg ccccaagacg cggtgtcgcg ctcgcaacgg cgaggtagaa
     1921 ctggcagggg taggagtggc atctacaggt ttgtgactcc aggagaacgg ccctcgggca
     1981 tgttcgattc ttcggtcctg tgtgagtgct atgacgcggg ctgtgcttgg tatgagctca
     2041 cgcccgctga gacctcggtt aggttgcggg cttacctaaa tacaccaggg ttgcccgtct
     2101 gccaggacca tctggagttc tgggagagcg tcttcacagg cctcacccac atagatgccc
     2161 acttcctgtc ccagactaaa caggcaggag acaactttcc ttacctggtg gcatatcaag
     2221 ctacagtgtg cgccagggct caagctccac ctccatcgtg ggaccaaatg tggaagtgtc
     2281 tcatacggct gaaacctaca ctgcacgggc caacacccct gctgtatagg ctaggagccg
     2341 tccaaaatga ggtcatcctc acacacccca taactaaata catcatggca tgcatgtcgg
     2401 ctgacctgga ggtcgtcact tctggtggtt ctactaatct gtcagatatt attgaaaagg
     2461 agaccggtaa gcaactggtt atccaggaat ccatcctcat gctcccagag gaggtggaag
     2521 aagtcattgg gaacaagccg gaaagcgata tactcgtgca caccgcctac gacgagagca
     2581 ccgacgagaa tgtcatgctt ctgactagcg acgcccctga atacaagcct tgggctctgg
     2641 tcatacagga tagcaacggt gagaacaaga ttaagatgct ctctggtggt tctcccaaga
     2701 agaagaggaa agtcgagggc agaggaagtc ttctaacatg cggtgacgtg gaggagaatc
     2761 ccggccctat ggtgtctaag ggcgaagagc tgattaagga gaacatgcac atgaagctgt
     2821 acatggaggg caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc
     2881 cctacgaggg cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg
     2941 ccttcgacat cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc
     3001 agggcatccc cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca
     3061 ccacatacga agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct
     3121 gcctcatcta caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc
     3181 agaagaaaac actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc
     3241 tggaaggcag aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaacg
     3301 ccaagaccac atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact
     3361 atgtggacta cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc
     3421 acgaggtggc agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatt
     3481 aaccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc
     3541 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     3601 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     3661 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     3721 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     3781 ctggggctcg ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     3841 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     3901 gtgtaaagcc tagggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     3961 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     4021 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     4081 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     4141 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     4201 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     4261 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     4321 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     4381 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     4441 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     4501 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     4561 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     4621 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     4681 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     4741 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     4801 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     4861 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     4921 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     4981 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     5041 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     5101 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     5161 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     5221 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     5281 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     5341 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     5401 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     5461 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     5521 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     5581 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     5641 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     5701 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     5761 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     5821 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     5881 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     5941 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     6001 tcgatctccc gatcccctag ggtcgactct cagtacaatc tgctctgatg ccgcatagtt
     6061 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     6121 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     6181 gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt gattattgac
     6241 tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg
     6301 cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt
     6361 gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca
     6421 atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt atc
//
