LOCUS       sequence-461183-        7265 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express HACE Editor as a fusion of AID cytidine deaminase and PcrA 
            helicase with mTagBFP2 reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7265)
  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 7265)
  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-PcrA-UGI-T2A-mTagBFP2
FEATURES             Location/Qualifiers
     source          1..7265
                     /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             3225..3473
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             3486..3506
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3507..3560
                     /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             3561..4271
                     /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             4280..4297
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4320..4337)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4326..4550
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4621..4637)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4621..4637)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4634..4656)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4645..4661
                     /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(4669..4675,4676..4693,4694..4699))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4714..4735
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4852..4869)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5023..5611)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5103..5122)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5782..6573,6574..6642))
                     /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     6405..6424
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6643..6747)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6826..6845)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7017..7265,1..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 gtatgaatgc cctgctgaac catatgaata cagaacaatc cgaggcggta aagaccacag
     1081 aaggcccctt gttgatcatg gcgggggctg ggagtggtaa aacgagggtc cttactcacc
     1141 gaatagcgta cttgcttgac gaaaaggacg tgagtccata taacgtgctt gccattacct
     1201 tcacaaacaa ggctgctaga gagatgaagg aaagagtcca aaaacttgtc ggtgaccaag
     1261 cggaggtcat ttggatgtct accttccatt ctatgtgcgt tcgcatactt cggcgagacg
     1321 cggataggat tgggatcgaa cggaacttca cgataataga tcctacagat caaaagtctg
     1381 taataaaaga tgttctcaaa aatgagaata tagatagcaa aaaatttgaa ccccgaatgt
     1441 tcataggtgc catatcaaac ttgaagaacg aactcaaaac acctgcggac gcacaaaagg
     1501 aagcaacaga ctaccacagt cagatggtcg caactgttta ttccggctac caacgacagc
     1561 tgagtcggaa tgaagcactg gattttgatg atctgatcat gactactatt aacctttttg
     1621 aaagagtacc ggaagtgttg gaatattacc aaaacaaatt tcaatatatc cacgttgatg
     1681 aataccaaga tactaataag gcacagtata cattggtaaa gctgctggcg tcaaagttta
     1741 aaaatctttg cgtggtcggg gatagtgacc agagcatata cggttggcgc ggcgccgaca
     1801 tacagaatat cttgtccttc gagaaagatt atcctgaggc gaatacaatc ttccttgagc
     1861 agaattatag atctacaaaa actattttga acgcggctaa cgaagtaata aaaaataata
     1921 gtgagcgaaa gcctaaaggt ctgtggacag ctaacacaaa tggtgaaaag attcattact
     1981 acgaagcaat gactgaacga gacgaagcgg agttcgtcat ccgggaaata atgaaacacc
     2041 aacgcaacgg caagaaatac caagacatgg caattctgta caggaccaat gcgcaatcca
     2101 gagttctcga agaaaccttt atgaagagca atatgccata cacgatggtt ggaggccaaa
     2161 aattctatga taggaaagag atcaaagacc tgctgagcta cctccgaatc attgccaaca
     2221 gtaacgacga catctcactt caacggatta ttaacgtacc gaaacgcggg gttggaccct
     2281 catcagttga gaaagttcaa aactatgcgt tgcagaacaa tatttccatg tttgacgctc
     2341 ttggagaagc tgattttatc ggcttgtcaa agaaagtaac ccaggagtgt cttaactttt
     2401 acgaactgat acaaagcctg ataaaggaac aggaattcct tgagatccac gagatcgtag
     2461 atgaagttct gcaaaaatcc ggctatcggg aaatgttgga aagggagaac acgctcgaaa
     2521 gtaggtcaag actcgagaac atagatgagt tcatgtcagt gcccaaagac tatgaggaga
     2581 atacgcccct tgaagagcag tcattgatca atttccttac tgacctgtca ctcgttgccg
     2641 atattgacga agctgacact gagaatgggg taacattgat gacgatgcac agtgctaagg
     2701 gattggagtt tcccatagtc ttcatcatgg gtatggagga gtccctcttt ccacacattc
     2761 gggcaatcaa atccgaggat gatcatgaga tgcaagagga gcgcagaatc tgttacgttg
     2821 cgattacacg agcggaggag gttctttata ttacacacgc aacgtctcgg atgctctttg
     2881 gacgcccaca gtcaaacatg ccctctaggt tccttaagga aatacccgag agcctgctgg
     2941 agaaccatag ctcaggtaag cggcagacca tacaaccaaa agctaagccg ttcgccaaac
     3001 gcggcttctc acagcgcacg actagcacga agaagcaggt gctctccagc gattggaatg
     3061 ttggcgataa ggttatgcac aaagcttggg gagagggcat ggtttccaat gtgaatgaga
     3121 aaaatggatc catagagctc gacatcatct ttaagagcca gggcccaaag cgcctgctcg
     3181 ctcagttcgc tccaatcgag aaaaaagaag actctggtgg ttctactaat ctgtcagata
     3241 ttattgaaaa ggagaccggt aagcaactgg ttatccagga atccatcctc atgctcccag
     3301 aggaggtgga agaagtcatt gggaacaagc cggaaagcga tatactcgtg cacaccgcct
     3361 acgacgagag caccgacgag aatgtcatgc ttctgactag cgacgcccct gaatacaagc
     3421 cttgggctct ggtcatacag gatagcaacg gtgagaacaa gattaagatg ctctctggtg
     3481 gttctcccaa gaagaagagg aaagtcgagg gcagaggaag tcttctaaca tgcggtgacg
     3541 tggaggagaa tcccggccct atggtgtcta agggcgaaga gctgattaag gagaacatgc
     3601 acatgaagct gtacatggag ggcaccgtgg acaaccatca cttcaagtgc acatccgagg
     3661 gcgaaggcaa gccctacgag ggcacccaga ccatgagaat caaggtggtc gagggcggcc
     3721 ctctcccctt cgccttcgac atcctggcta ctagcttcct ctacggcagc aagaccttca
     3781 tcaaccacac ccagggcatc cccgacttct tcaagcagtc cttccctgag ggcttcacat
     3841 gggagagagt caccacatac gaagacgggg gcgtgctgac cgctacccag gacaccagcc
     3901 tccaggacgg ctgcctcatc tacaacgtca agatcagagg ggtgaacttc acatccaacg
     3961 gccctgtgat gcagaagaaa acactcggct gggaggcctt caccgagacg ctgtaccccg
     4021 ctgacggcgg cctggaaggc agaaacgaca tggccctgaa gctcgtgggc gggagccatc
     4081 tgatcgcaaa cgccaagacc acatatagat ccaagaaacc cgctaagaac ctcaagatgc
     4141 ctggcgtcta ctatgtggac tacagactgg aaagaatcaa ggaggccaac aacgagacct
     4201 acgtcgagca gcacgaggtg gcagtggcca gatactgcga cctccctagc aaactggggc
     4261 acaagcttaa ttaaccggtc atcatcacca tcaccattga gtttaaaccc gctgatcagc
     4321 ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt
     4381 gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca
     4441 ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga
     4501 ggattgggaa gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc
     4561 ggaaagaacc agctggggct cgataccgtc gacctctagc tagagcttgg cgtaatcatg
     4621 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     4681 cggaagcata aagtgtaaag cctagggtgc ctaatgagtg agctaactca cattaattgc
     4741 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     4801 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     4861 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     4921 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     4981 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5041 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5101 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5161 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     5221 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     5281 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     5341 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     5401 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     5461 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     5521 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     5581 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     5641 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     5701 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     5761 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     5821 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     5881 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     5941 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     6001 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     6061 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     6121 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     6181 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     6241 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     6301 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     6361 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     6421 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     6481 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     6541 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     6601 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     6661 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     6721 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga
     6781 cggatcggga gatcgatctc ccgatcccct agggtcgact ctcagtacaa tctgctctga
     6841 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     6901 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
     6961 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
     7021 ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc atagcccata
     7081 tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga
     7141 cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt
     7201 ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt
     7261 gtatc
//
