LOCUS       sequence-441252-        7247 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Express HACE Editor as a fusion of AID cytidine deaminase and an 
            optimized PcrA M6 helicase with mTagBFP2 reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7247)
  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 7247)
  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 M6-UGI-T2A-mTagBFP2
FEATURES             Location/Qualifiers
     source          1..7247
                     /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             3207..3455
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             3468..3488
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             3489..3542
                     /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             3543..4253
                     /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             4262..4279
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4302..4319)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4308..4532
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4603..4619)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4603..4619)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4616..4638)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4627..4643
                     /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(4651..4657,4658..4675,4676..4681))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4696..4717
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4834..4851)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5005..5593)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5085..5104)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5764..6555,6556..6624))
                     /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     6387..6406
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6625..6729)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6808..6827)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6999..7247,1..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 gtatgaactt cctgtctgag caactgctgg cacacctgaa caaggagcag caagaagctg
     1081 tgcggaccac cgagggacct ctgctgatca tggccggcgc tggaagcgga aaaacaagag
     1141 tgctgacaca ccgcatcgcc tacctgatgg ctgagaagca cgtggcccct tggaacatcc
     1201 tggccattac ctttacaaac aaggccgcta gagagatgag agaaagagtg cagagcctcc
     1261 tgggaggcgc cgccgaggac gtgtggatca gcaccttcgc cagcatggcc gtgcggatcc
     1321 tgagaagaga tatcgacaga atcggcatca accggaactt cagcatcctg gacccaacag
     1381 accagctgag cgtgatgaaa accatcctga aagaaaagaa catcgacccc aagaaattcg
     1441 agcctagaac aatcctgggc acaatcagcg ccgccaagaa tgaactgctg cctccagaac
     1501 agtttgcgaa gcgggcctcc acctactatg agaaagtggt gtctgacgtc taccaggagt
     1561 atcagcagcg gctgctcagg tgtcacagcc ttgacttcga tgatctgatc atgaccacaa
     1621 tccagctgtt tgaccgggtc cccgacgtgc tccactacta ccaatacaag tttcagtaca
     1681 tccacatcga tgagtaccag gacacaaaca gagcccaata caccctggtg aaaaagctgg
     1741 ctgagcggtt ccagaacatc gccgccgtgg gcgacgccga tcagtctatc tacagatggc
     1801 ggggcgccga catccagaac atcctgagct tcgaaagaga ttaccccaac gccaaagtga
     1861 tcctgctgga gcaaaattac cggagcacga agcgcatcct gcaggccgca aacgaggtga
     1921 tcgagcacaa cgtgaacaga aagcctaagc ggatctggac cgagaatcct gagggcaagc
     1981 ccatcctgta ctacgaggcc atgaacgaag ccgacgaggc ccagttcgtg gccggcagaa
     2041 tcagagaggc cgtggagcgc ggcgagagaa gataccgaga cttcgccgtg ctgtacagaa
     2101 ccaatgccca gtccagagtc atggaagaga tgctgctgaa ggccaacatc ccttaccaga
     2161 tagtgggcgg cgtgaagttc tacgacagaa aggaaatcaa ggacatcctg gcttatctgc
     2221 gggtcatcgc aaacccagac gacgactgca gcctgctgag aattatcaac gttcctaaaa
     2281 gaggcatcgg agcctctacc atcgacaagc tggtccggta cgccgccgac cacgagctga
     2341 gcctgttcga ggccctgggc gagctggaga tgatcggtct tggcgccaag gccgctgggg
     2401 ccctggccgc cttcagaagc cagctggaac agtggacaca gctgcaggag tacgtgagcg
     2461 tgaccgaact ggtggaagag gtgctggaca agtctggcta ccgggaaatg ctgaaggccg
     2521 aaagaaccat cgaagcccag agcagactgg agaacctgga tgagttcctg agcgtgacca
     2581 agcatttcga aaacgtgtcc gacgacaaga gcctgatcgc cttcctgacc gatctggctc
     2641 tgatcagcga cctggacgag ctggatggca ccgagcaggc cgccgagggc gacgccgtga
     2701 tgctgatgac cctgcacgcc gccaagggcc tggaattccc cgtggtcttt ctgataggca
     2761 tggaagaagg catcttccct cacaatagaa gcctggagga tgacgatgag atggaagagg
     2821 agagaagact ggcctacgtg ggcatcacca gagctgaaga ggaactggtg ctgaccagcg
     2881 cccagatgcg gaccctgttc ggcaacattc agatggaccc tcctagccgg ttccttaacg
     2941 agatccccgc ccatctgctt gagacagcca gcagacggca ggccggagct tccaggcctg
     3001 ccgtgagcag accccaggcc tctggcgctg tgggcagctg gaaggtgggc gatcgggcaa
     3061 atcacagaaa gtggggcatc ggaaccgtgg tttccgtgcg gggcggcgga gatgatcagg
     3121 aactggacat cgcttttcca tctcctatcg gcattaagag actgctagct aaattcgccc
     3181 ctatcgagaa ggtgtctggt ggttctacta atctgtcaga tattattgaa aaggagaccg
     3241 gtaagcaact ggttatccag gaatccatcc tcatgctccc agaggaggtg gaagaagtca
     3301 ttgggaacaa gccggaaagc gatatactcg tgcacaccgc ctacgacgag agcaccgacg
     3361 agaatgtcat gcttctgact agcgacgccc ctgaatacaa gccttgggct ctggtcatac
     3421 aggatagcaa cggtgagaac aagattaaga tgctctctgg tggttctccc aagaagaaga
     3481 ggaaagtcga gggcagagga agtcttctaa catgcggtga cgtggaggag aatcccggcc
     3541 ctatggtgtc taagggcgaa gagctgatta aggagaacat gcacatgaag ctgtacatgg
     3601 agggcaccgt ggacaaccat cacttcaagt gcacatccga gggcgaaggc aagccctacg
     3661 agggcaccca gaccatgaga atcaaggtgg tcgagggcgg ccctctcccc ttcgccttcg
     3721 acatcctggc tactagcttc ctctacggca gcaagacctt catcaaccac acccagggca
     3781 tccccgactt cttcaagcag tccttccctg agggcttcac atgggagaga gtcaccacat
     3841 acgaagacgg gggcgtgctg accgctaccc aggacaccag cctccaggac ggctgcctca
     3901 tctacaacgt caagatcaga ggggtgaact tcacatccaa cggccctgtg atgcagaaga
     3961 aaacactcgg ctgggaggcc ttcaccgaga cgctgtaccc cgctgacggc ggcctggaag
     4021 gcagaaacga catggccctg aagctcgtgg gcgggagcca tctgatcgca aacgccaaga
     4081 ccacatatag atccaagaaa cccgctaaga acctcaagat gcctggcgtc tactatgtgg
     4141 actacagact ggaaagaatc aaggaggcca acaacgagac ctacgtcgag cagcacgagg
     4201 tggcagtggc cagatactgc gacctcccta gcaaactggg gcacaagctt aattaaccgg
     4261 tcatcatcac catcaccatt gagtttaaac ccgctgatca gcctcgactg tgccttctag
     4321 ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac
     4381 tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga gtaggtgtca
     4441 ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg aagacaatag
     4501 caggcatgct ggggatgcgg tgggctctat ggcttctgag gcggaaagaa ccagctgggg
     4561 ctcgataccg tcgacctcta gctagagctt ggcgtaatca tggtcatagc tgtttcctgt
     4621 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     4681 agcctagggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     4741 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     4801 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     4861 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     4921 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4981 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     5041 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5101 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5161 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     5221 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5281 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5341 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5401 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5461 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5521 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5581 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     5641 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     5701 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     5761 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     5821 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     5881 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     5941 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     6001 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     6061 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     6121 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     6181 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     6241 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     6301 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     6361 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     6421 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     6481 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     6541 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     6601 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     6661 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     6721 ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc gacggatcgg gagatcgatc
     6781 tcccgatccc ctagggtcga ctctcagtac aatctgctct gatgccgcat agttaagcca
     6841 gtatctgctc cctgcttgtg tgttggaggt cgctgagtag tgcgcgagca aaatttaagc
     6901 tacaacaagg caaggcttga ccgacaattg catgaagaat ctgcttaggg ttaggcgttt
     6961 tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta
     7021 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
     7081 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
     7141 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
     7201 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatc
//
