LOCUS       sequence-437082-        9326 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express HACE Editor as a fusion of AID cytidine deaminase and BLM 
            helicase with mTagBFP2 reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9326)
  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 9326)
  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-BLM-UGI-T2A-mTagBFP2
FEATURES             Location/Qualifiers
     source          1..9326
                     /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             5286..5534
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5547..5567
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5568..5621
                     /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             5622..6332
                     /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             6341..6358
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(6381..6398)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    6387..6611
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(6682..6698)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6682..6698)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6695..6717)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6706..6722
                     /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(6730..6736,6737..6754,6755..6760))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6775..6796
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6913..6930)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7084..7672)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7164..7183)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7843..8634,8635..8703))
                     /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     8466..8485
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8704..8808)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8887..8906)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(9078..9326,1..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 gtatggctgc tgttcctcaa aataatctac aggagcaact agaacgtcac tcagccagaa
     1081 cacttaataa taaattaagt ctttcaaaac caaaattttc aggtttcact tttaaaaaga
     1141 aaacatcttc agataacaat gtatctgtaa ctaatgtgtc agtagcaaaa acacctgtat
     1201 taagaaataa agatgttaat gttaccgaag acttttcctt cagtgaacct ctacccaaca
     1261 ccacaaatca gcaaagggtc aaggacttct ttaaaaatgc tccagcagga caggaaacac
     1321 agagaggtgg atcaaaatca ttattgccag atttcttgca gactccgaag gaagttgtat
     1381 gcactaccca aaacacacca actgtaaaga aatcccggga tactgctctc aagaaattag
     1441 aatttagttc ttcaccagat tctttaagta ccatcaatga ttgggatgat atggatgact
     1501 ttgatacttc tgagacttca aaatcatttg ttacaccacc ccaaagtcac tttgtaagag
     1561 taagcactgc tcagaaatca aaaaagggta agagaaactt ttttaaagca cagctttata
     1621 caacaaacac agtaaagact gacttgcctc caccctcctc tgaaagcgag caaatagatt
     1681 tgactgagga acagaaggat gactcagaat ggttaagcag cgatgtgatt tgcatcgatg
     1741 atggccccat tgctgaagtg catataaatg aagatgctca ggaaagtgac tctctgaaaa
     1801 ctcatttgga agatgaaaga gataatagcg aaaagaagaa gaatttggaa gaagctgaat
     1861 tacattcaac tgagaaagtt ccatgtattg aatttgatga tgatgattat gatacggatt
     1921 ttgttccacc ttctccagaa gaaattattt ctgcttcttc ttcctcttca aaatgcctta
     1981 gtacgttaaa ggaccttgac acctctgaca gaaaagagga tgttcttagc acatcaaaag
     2041 atcttttgtc aaaacctgag aaaatgagta tgcaggagct gaatccagaa accagcacag
     2101 actgtgacgc tagacagata agtttacagc agcagcttat tcatgtgatg gagcacatct
     2161 gtaaattaat tgatactatt cctgatgata aactgaaact tttggattgt gggaacgaac
     2221 tgcttcagca gcggaacata agaaggaaac ttctaacgga agtagatttt aataaaagtg
     2281 atgccagtct tcttggctca ttgtggagat acaggcctga ttcacttgat ggccctatgg
     2341 agggtgattc ctgccctaca gggaattcta tgaaggagtt aaatttttca caccttccct
     2401 caaattctgt ttctcctggg gactgtttac tgactaccac cctaggaaag acaggattct
     2461 ctgccaccag gaagaatctt tttgaaaggc ctttattcaa tacccattta cagaagtcct
     2521 ttgtaagtag caactgggct gaaacaccaa gactaggaaa aaaaaatgaa agctcttatt
     2581 tcccaggaaa tgttctcaca agcactgctg tgaaagatca gaataaacat actgcttcaa
     2641 taaatgactt agaaagagaa acccaacctt cctatgatat tgataatttt gacatagatg
     2701 actttgatga tgatgatgac tgggaagaca taatgcataa tttagcagcc agcaaatctt
     2761 ccacagctgc ctatcaaccc atcaaggaag gtcggccaat taaatcagta tcagaaagac
     2821 tttcctcagc caagacagac tgtcttccag tgtcatctac tgctcaaaat ataaacttct
     2881 cagagtcaat tcagaattat actgacaagt cagcacaaaa tttagcatcc agaaatctga
     2941 aacatgagcg tttccaaagt cttagttttc ctcatacaaa ggaaatgatg aagatttttc
     3001 ataaaaaatt tggcctgcat aattttagaa ctaatcagct agaggcgatc aatgctgcac
     3061 tgcttggtga agactgtttt atcctgatgc cgactggagg tggtaagagt ttgtgttacc
     3121 agctccctgc ctgtgtttct cctggggtca ctgttgtcat ttctcccttg agatcactta
     3181 tcgtagatca agtccaaaag ctgacttcct tggatattcc agctacatat ctgacaggtg
     3241 ataagactga ctcagaagct acaaatattt acctccagtt atcaaaaaaa gacccaatca
     3301 taaaactcct atatgtcact ccagaaaaga tctgtgcaag taacagactc atttctactc
     3361 tggagaatct ctatgagagg aagctcttgg cacgttttgt tattgatgaa gcacattgtg
     3421 tcagtcagtg gggacatgat tttcgtcaag attacaaaag aatgaatatg cttcgccaga
     3481 agtttccttc tgttccggtg atggctctta cggccacagc taatcccagg gtacagaagg
     3541 acatcctgac tcagctgaag attctcagac ctcaggtgtt tagcatgagc tttaacagac
     3601 ataatctgaa atactatgta ttaccgaaaa agcctaaaaa ggtggcattt gattgcctag
     3661 aatggatcag aaagcaccac ccatatgatt cagggataat ttactgcctc tccaggcgag
     3721 aatgtgacac catggctgac acgttacaga gagatgggct cgctgctctt gcttaccatg
     3781 ctggcctcag tgattctgcc agagatgaag tgcagcagaa gtggattaat caggatggct
     3841 gtcaggttat ctgtgctaca attgcatttg gaatggggat tgacaaaccg gacgtgcgat
     3901 ttgtgattca tgcatctctc cctaaatctg tggagggtta ctaccaagaa tctggcagag
     3961 ctggaagaga tggggaaata tctcactgcc tgcttttcta tacctatcat gatgtgacca
     4021 gactgaaaag acttataatg atggaaaaag atggaaacca tcatacaaga gaaactcact
     4081 tcaataattt gtatagcatg gtacattact gtgaaaatat aacagaatgc aggagaatac
     4141 agcttttggc ctactttggt gaaaatggat ttaatcctga tttttgtaag aaacacccag
     4201 atgtttcttg tgataattgc tgtaaaacaa aggattataa aacaagagat gtgactgacg
     4261 atgtgaaaag tattgtaaga tttgttcaag aacatagttc atcacaagga atgagaaata
     4321 taaaacatgt aggtccttct ggaagattta ctatgaatat gctggtcgac attttcttgg
     4381 ggagtaagag tgcaaaaatc cagtcaggta tatttggaaa aggatctgct tattcacgac
     4441 acaatgccga aagacttttt aaaaagctga tacttgacaa gattttggat gaagacttat
     4501 atatcaatgc caatgaccag gcgatcgctt atgtgatgct cggaaataaa gcacaaactg
     4561 tactaaatgg caatttaaag gtagacttta tggaaacaga aaattccagc agtgtgaaaa
     4621 aacaaaaagc gttagtagca aaagtgtctc agagggaaga gatggttaaa aaatgtcttg
     4681 gagaacttac agaagtctgc aaatctctgg ggaaagtttt tggtgtccat tacttcaata
     4741 tttttaatac cgtcactctc aagaagcttg cagaatcttt atcttctgat cctgaggttt
     4801 tgcttcaaat tgatggtgtt actgaagaca aactggaaaa atatggtgcg gaagtgattt
     4861 cagtattaca gaaatactct gaatggacat cgccagctga agacagttcc ccagggataa
     4921 gcctgtccag cagcagaggc cccggaagaa gtgccgctga ggagcttgac gaggaaatac
     4981 ccgtatcttc ccactacttt gcaagtaaaa ccagaaatga aaggaagagg aaaaagatgc
     5041 cagcctccca aaggtctaag aggagaaaaa ctgcttccag tggttccaag gcaaaggggg
     5101 ggtctgccac atgtagaaag atatcttcca aaacgaaatc ctccagcatc attggatcca
     5161 gttcagcctc acatacttct caagcgacat caggagccaa tagcaaattg gggattatgg
     5221 ctccaccgaa gcctataaat agaccgtttc ttaagccttc atatgcattc tcatctggtg
     5281 gttctactaa tctgtcagat attattgaaa aggagaccgg taagcaactg gttatccagg
     5341 aatccatcct catgctccca gaggaggtgg aagaagtcat tgggaacaag ccggaaagcg
     5401 atatactcgt gcacaccgcc tacgacgaga gcaccgacga gaatgtcatg cttctgacta
     5461 gcgacgcccc tgaatacaag ccttgggctc tggtcataca ggatagcaac ggtgagaaca
     5521 agattaagat gctctctggt ggttctccca agaagaagag gaaagtcgag ggcagaggaa
     5581 gtcttctaac atgcggtgac gtggaggaga atcccggccc tatggtgtct aagggcgaag
     5641 agctgattaa ggagaacatg cacatgaagc tgtacatgga gggcaccgtg gacaaccatc
     5701 acttcaagtg cacatccgag ggcgaaggca agccctacga gggcacccag accatgagaa
     5761 tcaaggtggt cgagggcggc cctctcccct tcgccttcga catcctggct actagcttcc
     5821 tctacggcag caagaccttc atcaaccaca cccagggcat ccccgacttc ttcaagcagt
     5881 ccttccctga gggcttcaca tgggagagag tcaccacata cgaagacggg ggcgtgctga
     5941 ccgctaccca ggacaccagc ctccaggacg gctgcctcat ctacaacgtc aagatcagag
     6001 gggtgaactt cacatccaac ggccctgtga tgcagaagaa aacactcggc tgggaggcct
     6061 tcaccgagac gctgtacccc gctgacggcg gcctggaagg cagaaacgac atggccctga
     6121 agctcgtggg cgggagccat ctgatcgcaa acgccaagac cacatataga tccaagaaac
     6181 ccgctaagaa cctcaagatg cctggcgtct actatgtgga ctacagactg gaaagaatca
     6241 aggaggccaa caacgagacc tacgtcgagc agcacgaggt ggcagtggcc agatactgcg
     6301 acctccctag caaactgggg cacaagctta attaaccggt catcatcacc atcaccattg
     6361 agtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     6421 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     6481 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     6541 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     6601 gggctctatg gcttctgagg cggaaagaac cagctggggc tcgataccgt cgacctctag
     6661 ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     6721 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctagggtg cctaatgagt
     6781 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     6841 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     6901 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     6961 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     7021 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     7081 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     7141 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     7201 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     7261 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     7321 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     7381 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     7441 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     7501 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     7561 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     7621 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     7681 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     7741 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     7801 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     7861 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     7921 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     7981 gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     8041 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     8101 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     8161 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     8221 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     8281 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     8341 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     8401 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     8461 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     8521 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     8581 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     8641 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     8701 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     8761 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     8821 aaaagtgcca cctgacgtcg acggatcggg agatcgatct cccgatcccc tagggtcgac
     8881 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
     8941 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
     9001 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
     9061 ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg
     9121 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
     9181 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
     9241 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
     9301 ccacttggca gtacatcaag tgtatc
//
