LOCUS       sequence-437077-        6500 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express HACE Editor as a fusion of AID cytidine deaminase and PcrA 
            helicase..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6500)
  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 6500)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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
FEATURES             Location/Qualifiers
     source          1..6500
                     /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             3515..3532
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3555..3572)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3561..3785
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3856..3872)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3856..3872)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3869..3891)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3880..3896
                     /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(3904..3910,3911..3928,3929..3934))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3949..3970
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4087..4104)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4258..4846)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4338..4357)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5017..5808,5809..5877))
                     /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     5640..5659
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5878..5982)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6061..6080)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6252..6500,1..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 aaagtctaac cggtcatcat caccatcacc attgagttta
     3541 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     3601 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     3661 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     3721 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     3781 tatggcttct gaggcggaaa gaaccagctg gggctcgata ccgtcgacct ctagctagag
     3841 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     3901 acacaacata cgagccggaa gcataaagtg taaagcctag ggtgcctaat gagtgagcta
     3961 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4021 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     4081 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     4141 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     4201 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     4261 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     4321 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     4381 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     4441 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     4501 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     4561 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     4621 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     4681 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4741 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4801 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4861 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4921 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     4981 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     5041 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     5101 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     5161 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     5221 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     5281 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     5341 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     5401 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     5461 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     5521 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     5581 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     5641 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     5701 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     5761 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     5821 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     5881 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     5941 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     6001 gccacctgac gtcgacggat cgggagatcg atctcccgat cccctagggt cgactctcag
     6061 tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt gtgtgttgga
     6121 ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct tgaccgacaa
     6181 ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg tacgggccag
     6241 atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
     6301 agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg
     6361 ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
     6421 gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
     6481 ggcagtacat caagtgtatc
//
