LOCUS       sequence-486091-        7731 bp    DNA     circular SYN 16-SEP-2025
DEFINITION  Mammalian expression of Flex-Cas12a adenine base editor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7731)
  AUTHORS   Ma E, Chen K, Shi H, Wasko KM, Esain-Garcia I, Trinidad MI, Zhou K, 
            Ye J, Doudna JA
  TITLE     Directed evolution expands CRISPR-Cas12a genome-editing capacity.
  JOURNAL   Nucleic Acids Res. 2025 Jul 8;53(13):gkaf649. doi: 
            10.1093/nar/gkaf649.
  PUBMED    40671523
REFERENCE   2  (bases 1 to 7731)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 16, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nucleic 
            Acids Res."; date: "2025-07-8"; volume: "53(13):gkaf649. doi"; 
            pages: " 10.1093/nar/gkaf649"
COMMENT     Sequence Label: pHS641_Flex-Cas12a-ABE
FEATURES             Location/Qualifiers
     source          1..7731
                     /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"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             445..465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4747..4767
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     primer_bind     complement(4786..4803)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4792..5016
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5087..5103)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5087..5103)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5100..5122)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5111..5127
                     /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(5135..5141,5142..5159,5160..5165))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5180..5201
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5318..5335)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5489..6077)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5569..5588)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6248..7039,7040..7108))
                     /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     6871..6890
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7109..7213)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7292..7311)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(7483..7731,1..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 ggccgctaat acgactcact atagggagag ccgccaccat gaaacggaca
      421 gccgacggaa gcgagttcga gtcaccaaag aagaagcgga aagtctctga ggtggagttt
      481 tcccacgagt actggatgag acatgccctg accctggcca agagggcacg cgatgagagg
      541 gaggtgcctg tgggagccgt gctggtgctg aacaatagag tgatcggcga gggctggaac
      601 agagccatcg gcctgcacga cccaacagcc catgccgaaa ttatggccct gagacagggc
      661 ggcctggtca tgcagaacta cagactgatt gacgccaccc tgtacgtgac attcgagcct
      721 tgcgtgatgt gcgccggcgc catgatccac tctaggatcg gccgcgtggt gtttggcgtg
      781 aggaactcaa aaagaggcgc cgcaggctcc ctgatgaacg tgctgaacta ccccggcatg
      841 aatcaccgcg tcgaaattac cgagggaatc ctggcagatg aatgtgccgc cctgctgtgc
      901 gatttctatc ggatgcctag acaggtgttc aatgctcaga agaaggccca gagctccatc
      961 aactctggtg gttcttctgg tggttctagc ggcagcgaga ctcccgggac ctcagagtcc
     1021 gccacacccg aaagttccgg agggagtagc ggcgggtcta gcaagctgga aaaatttacc
     1081 aactgttaca gcctgagcaa aaccctgagg ttcaaggcca tccccgtggg caagacccaa
     1141 gagaacatcg ataacaagag actgctggtc gaggatgaga agcgtgcaga ggactacaag
     1201 ggcgtgaaga agctcctgga tcggtactac ctgagtttca tcaacgacgt gctgcacagc
     1261 atcaagctga agaacctgaa caactacatc agcctgttta gaaagaaaac cagaaccgaa
     1321 aaagaaaata aggagctgga aaatctggaa atcaacctga gaaaggaaat cgctaaggcc
     1381 tttaagggaa acgaaggata caaatccctg tttaagaaag acatcatcga gacaattctg
     1441 ccagagttcc tggacgacaa agacgagata gctcttgtga acagcttcaa caggttcacg
     1501 accgccttca ccggattctt cgacaacagg gaaaacatgt tcagtgagga agccaagtct
     1561 accagcatcg ctttccggtg catcaatgag aacctgaccg tgtatatctc taatatggat
     1621 atctttgaaa aagtggacgc tattttcgac aagcacgagg tgcaggagat caaggagaag
     1681 atcctgaaca gcgactacga cgtggaggac ttcttcgagg gcgaattttt caacttcgtg
     1741 ctgacacaag agggcatcga cgtttacaac gccatcatag gcggcttcgt gaccgagtcc
     1801 ggcgaaaaga tcaagggcct taacgagtac atcaacctgt ataaccagaa aaccaagcag
     1861 aagctgccta agttcaagcc tctttataag caggtcctga gcgaccggga aagcctgagc
     1921 ttctacggcg agggctacac atctgacgag gaagtgctgg aggtctttcg gaataccctg
     1981 aacaagaact ctgagatctt cagctctatt aagaaactgg aaaagctgtt caagaatttc
     2041 gacgagtata gtagcgccgg gatcttcgtg aagaacggcc ctgccattag caccatcagc
     2101 aaggacatct tcggcgaatg gaacgttatc cgcgataagt ggaacgccga gtacgatgac
     2161 atccacctga aaaagaaggc cgtggtgacc gagaagtacg aagatgacag aagaaagagc
     2221 ttcaaaaaga tcggctcatt tagcctggaa cagctgcagg agtacgccga cgccgacctg
     2281 tccgtggtcg aaaagctgaa ggaaattatc atccaaaaag tggacgagat ctataaggtg
     2341 tacgggagct ctgagaagct gttcgacgcc gactttgtgc tggaaaagtc actgaaaaag
     2401 aacgacgctg tggtcgccat catgaaggat ctgctggact ccgtgaagag cttcgagaac
     2461 tacatcaagg ccttcttcgg cgaaggcaag gagaccaacc gggacgagtc tttttacggc
     2521 gatttcgtgc tggcctacga catcctgctg aaggtggacc acatctacga cgccatcaga
     2581 aactacgtga cccagaagcc ctacagcaaa gacaagttta agctgtactt ccagaatcct
     2641 cagttcatgg gaggatgggg caaagacaag gaaaccgact accgggctac aattctgaga
     2701 tacggcttta agtactacct tgcaatcatg gacaaaaagt atgccaagtg cctgcagaaa
     2761 attgataagg atgacgtgaa cggcaactac gagaagatca actacaagct cctgcccggc
     2821 cccaacaaga tgctgccaaa ggtgttcttc tccaaaaagt ggatggccta ttacaaccct
     2881 tctgaggata tccagaagat ctataagaac ggaacattca agaagggcga tatgttcaac
     2941 ctgaatgact gccacaagct gatcgacttc tttaaagaca gcatctccag ataccctaag
     3001 tggtccaacg cctacgactt caacttcagc gaaacagaaa agtacaagaa catcgccggc
     3061 ttctacagag aggtggaaga acaaggctac aaggtcagct tcgagagcgc tagcaagaag
     3121 gaggtggaca agctggtgga ggagggaaag ctgtacatgt tccagatcta caacaaggat
     3181 ttcagcgaca agagccacgg cacacctaac ctgcacacca tgtatttcaa gctgctgttt
     3241 gacgagaaca accatggcca gatccggctg tctggcggcg ccgaactgtt catgcggaga
     3301 gcctctttga agaaggaaga gctggtggtg caccctgcca atagccctat cgccaacaag
     3361 aacccagata acccaaagaa aaccaccaca ctgagctacg acgtgtacaa agataagaga
     3421 ttcagcgagg atcagtacca gctgcatatc cctatcgcca tcaataagtg tcctaaaaac
     3481 atcttcaaga tcaataccga ggtgcgggtg ttgctgaagc acgacgataa cccttacgtg
     3541 atcggaatcg ccaggggcga gagaaacctg ctgtacatcg tggtggtgga cggcaagggc
     3601 aatatcgtgg agcaatacag cctgaatgag atcatcaaca atttcaacgg catccgcata
     3661 aaaacagatt accactctct gctggataag aaagagaaag agcggtttga ggccagacag
     3721 aactggacca gcatcgagaa tatcaaagag ttaaaggccg gctacatcag ccaggtggtg
     3781 cacaaaatct gcgagctggt ggaaaagtac gacgctgtga tcgcccttga ggatctaaac
     3841 agcggcttta agaacagcag agtgaaggta gaaaagcagg tgtatcagaa gttcgagaag
     3901 atgctcatcg ataagctgaa ctacatggtg gataagaaaa gcaacccctg cgccacaggc
     3961 ggagctctga agggctacca gatcaccaac aaattcgaga gctttaagag catgtcgacc
     4021 cagaatggtt tcatctttta catccccgcc tggctcacct caaagatcga tcccagcaca
     4081 ggcttcgtca acctgctgaa aaccaagtac accagcatcg ccgatagcaa gaagtttatc
     4141 tcgtccttcg acagaatcat gtacgtgccc gaggaggacc tgttcgagtt cgccctggac
     4201 tacaagaact tctcccggac agatgccgac tacattaaga agtggaagct gtacagctac
     4261 ggaaacagga tcagaatctt tagaaacccc aaaaagaaca acgtattcga ttgggaggaa
     4321 gtgtgcctga cctcggccta caaggagctg ttcaacaagt acggcatcaa ctaccagcag
     4381 ggcgacatcc gggccctgct ctgtgaacag agcgacaagg ctttctacag cagcttcatg
     4441 gccctgatga gcctgatgct gcagatgaga aacagcatca caggaagaac agacgtggac
     4501 tttctgatct ctcctgtgaa gaacagcgac ggcatcttct acgacagcag aaactatgaa
     4561 gcccaggaga acgcgatcct gcctaagaac gcagacgcca acggcgccta caatatcgcc
     4621 agaaaggtgc tgtgggccat tggccagttc aagaaggctg aagatgagaa actggataaa
     4681 gtgaagatcg ccatctccaa caaggaatgg ctggaatacg cccagaccag cgtgaagcac
     4741 ggatccccca agaagaagag gaaagtctaa aaacccgctg atcagcctcg actgtgcctt
     4801 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     4861 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     4921 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     4981 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     5041 ggggctcgat accgtcgacc tctagctaga gcttggcgta atcatggtca tagctgtttc
     5101 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     5161 gtaaagccta gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     5221 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     5281 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     5341 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     5401 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     5461 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     5521 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     5581 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     5641 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     5701 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     5761 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     5821 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     5881 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     5941 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     6001 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     6061 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac actcagtgga
     6121 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     6181 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     6241 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     6301 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     6361 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     6421 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     6481 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     6541 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     6601 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     6661 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     6721 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     6781 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     6841 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     6901 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     6961 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     7021 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     7081 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     7141 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     7201 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtcgacgga tcgggagatc
     7261 gatctcccga tcccctaggg tcgactctca gtacaatctg ctctgatgcc gcatagttaa
     7321 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     7381 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     7441 gttttgcgct gcttcgcgat gtacgggcca gatatacgcg ttgacattga ttattgacta
     7501 gttattaata gtaatcaatt acggggtcat tagttcatag cccatatatg gagttccgcg
     7561 ttacataact tacggtaaat ggcccgcctg gctgaccgcc caacgacccc cgcccattga
     7621 cgtcaataat gacgtatgtt cccatagtaa cgccaatagg gactttccat tgacgtcaat
     7681 gggtggagta tttacggtaa actgcccact tggcagtaca tcaagtgtat c
//
