LOCUS       sequence-365709-        7132 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Directed evolution of Cas9 adenine base editors with altered PAM 
            compatibility.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7132)
  AUTHORS   Huang TP, Heins ZJ, Miller SM, Wong BG, Balivada PA, Wang T, Khalil 
            AS, Liu DR
  TITLE     High-throughput continuous evolution of compact Cas9 variants 
            targeting single-nucleotide-pyrimidine PAMs.
  JOURNAL   Nat Biotechnol. 2022 Sep 8. pii: 10.1038/s41587-022-01410-2. doi: 
            10.1038/s41587-022-01410-2.
  PUBMED    36076084
REFERENCE   2  (bases 1 to 7132)
  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: "Nat 
            Biotechnol. 2022 Sep 8. pii: 10.1038/s41587-022-01410-2. doi: 
            10.1038/s41587-022-01410-2."
COMMENT     Sequence Label: pTPH405c
FEATURES             Location/Qualifiers
     source          1..7132
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      3..591
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     492..511
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     745..762
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    777..917
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(843..862)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1003..1025
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1019..1210)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     terminator      1321..1367
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             3402..4481
                     /codon_start=1
                     /gene="luxA"
                     /product="LuxA luciferase subunit"
                     /label=LuxA
                     /note="from the Photorhabdus luminescens bioluminescence 
                     operon"
                     /translation="MKFGNFLLTYQPPQFSQTEVMKRLVKLGRISEECGFDTVWLLEHH
                     FTEFGLLGNPYVAAAYLLGATKKLNVGTAAIVLPTAHPVRQLEDVNLLDQMSKGRFRFG
                     ICRGLYNKDFRVFGTDMNNSRALAECWYGLIKNGMTEGYMEADNEHIKFHKVKVNPAAY
                     SRGGAPVYVVAESASTTEWAAQFGLPMILSWIINTNEKKAQLELYNEVAQEYGHDIHNI
                     DHCLSYITSVDHDSIKAKEICRKFLGHWYDSYVNATTIFDDSDQTRGYDFNKGQWRDFV
                     LKGHKDTNRRIDYSYEINPVGTPQECIDIIQKDIDATGISNICCGFEANGTVDEIIASM
                     KLFQSDVMPFLKKKQRSLLY"
     CDS             4545..5525
                     /codon_start=1
                     /gene="luxB"
                     /product="LuxB luciferase subunit"
                     /label=LuxB
                     /note="from the Photorhabdus luminescens bioluminescence 
                     operon"
                     /translation="KFGLFFLNFINSTTVQEQSIVRMQEITEYVDKLNFEQILVYENHF
                     SDNGVVGAPLTVSGFLLGLTEKIKIGSLNHIITTHHPVRIAEEACLLDQLSEGRFILGF
                     SDCEKKDEMHFFNRPVEYQQQLFEECYEIINDALTTGYCNPDNDFYSFPKISVNPHAYT
                     PGGPRKYVTATSHHIVEWAAKKGIPLIFKWDDSNDVRYEYAERYKAVADKYDVDLSEID
                     HQLMILVNYNEDSNKAKQETRAFISDYVLEMHPNENFENKLEEIIAENAVGNYTECITA
                     AKLAIEKCGAKSVLLSFEPMNDLMSQKNVINIVDDNIKKYHTEYT"
     misc_RNA        complement(5638..5730)
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     promoter        complement(join(5787..5793,5794..5811,5812..5817))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5832..5853
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(join(6065..6856,6857..6925))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHFTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6688..6707
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6926..7030)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7098..7116
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cgttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
       61 ccagcggtgg tttgtttgcc ggatcaagag ctaccacctc tttttccgaa ggtaactggc
      121 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
      181 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
      241 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
      301 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
      361 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
      421 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      481 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      541 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      601 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      661 gcgttatccc ctgattctgt ggataaccgt attacagcct ttgagtgagc tgataccgct
      721 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
      781 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc
      841 agtacaatct gctctgatgc cgcatagtta agccagtata cactccgcta tcgctacgtg
      901 actgggtcat ggctgcgccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
      961 gtcttctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     1021 agaggttttc accgtcatca ccgaaacgcg cgaggcagct gcggtaaagc tcatcagcgt
     1081 ggtcgtgaag cgattcacag atgtctgcct gttcatccgc gtccagctcg ttgagtttct
     1141 ccagaagcgt taatgtctgg cttctgataa agcgggccat gttaagggcg gttttttcct
     1201 gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt catgggggta atgataccga
     1261 tgaaacgaga gaggatgctc acgatacggg ttactgatga tgaacaagag gacatccggt
     1321 caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatgga aagaagagaa
     1381 accgggactg gttcgttttg ctgaggagac ttagggactc tcacagctaa caccacgtcg
     1441 tccctatctg ctgccctagg tctatgagtg gttgctggat aactttacgg gcatgcataa
     1501 ggctcgtatg atatattcag ggagaccaca acggtttccc tctacaaata attttgttta
     1561 acttttacta gagtgggacc ctacctgcag gtgcagtaaa ggaaaaaaaa aatgaaaaaa
     1621 ttattatttg cgataccgct gtgcttaagc tacgaaactg aaatcttgac cgtcgaatat
     1681 ggtctgctgc caatcggcaa gattgttgaa aaacgtattg aatgtacggt ctactcagtg
     1741 gataacaacg gcaatatcta cacccagccg gtggcccagt ggcatgaccg tggtgaacag
     1801 gaagtgttcg aatattgtct ggaagacgga tctttaatcc gtgccacaaa ggatcacaaa
     1861 tttatgactg tagatggtca gatgctccca atcgacgaaa tttttgaacg cgaattagac
     1921 ctgatgcgcg tggataatct cccgaattcc ggaggatcta gcggggcaac ctctggctct
     1981 gagtagtgag gaacaagcga gagtgcaaca cctgaaagca gcgggggcag cagcgggggg
     2041 tcaatcaaaa ttgccacgcg taaatattta ggcaaacaga atgtttatga tatcggtgtc
     2101 gagcgcgatc ataatttcgc gctgaaaaac ggatttatag ctagcaattg tttcaacgtt
     2161 gttcctttct attctcactc cgccgaaact gttgaaagtt gtttagcaaa accccataca
     2221 gaaaattcat ttactaacgt ctggaaagac gacaaaactt tagatcgtta cgctaactat
     2281 gagggctgtc tgtggaatgc tacaggcgtt gtagtttgta ctggtgacga aactcagtgt
     2341 tacggtacat gggttcctat tgggcttgct atccctgaaa atgagggtgg tggctctgag
     2401 ggtggcggtt ctgagggtgg cggttctgag ggtggtggta ctaaacctcc tgagtacggt
     2461 gatacaccta ttccgggcta tacttatatc aaccctctcg acggcactta tccgcctggt
     2521 actgagcaaa accccgctaa tcctaatcct tctcttgagg agtctcagcc tcttaatact
     2581 ttcatgtttc agaataatag gttccgaaat aggcaggggg cattaactgt ttatacgggc
     2641 actgttactc aaggcactga ccccgttaaa acttattacc agtacactcc tgtatcatca
     2701 aaagccatgt atgacgctta ctggaacggt aaattcagag actgcgcttt ccattctggc
     2761 tttaatgagg atccattcgt ttgtgaatat caaggccaat cgtctgacct gcctcaacct
     2821 cctgtcaatg ctggcggcgg ctctggtggt ggttctggtg gcggctctga gggtggtggc
     2881 tctgagggtg gcggttctga gggtggcggc tctgagggag gcggttccgg tggtggctct
     2941 ggttccggtg attttgatta tgaaaagatg gcaaacgcta ataagggggc tatgaccgaa
     3001 aatgccgatg aaaacgcgct acagtctgac gctaaaggca aacttgattc tgtcgctact
     3061 gattacggtg ctgctatcga tggtttcatt ggtgacgttt ccggccttgc taatggtaat
     3121 ggtgctactg gtgattttgc tggctctaat tcccaaatgg ctcaagtcgg tgacggtgat
     3181 aattcacctt taatgaataa tttccgtcaa tatttacctt ccctccctca atcggttgaa
     3241 tgtcgccctt ttgtctttgg cgctggtaaa ccttacgagt tcagtatcga ctgcgataag
     3301 atcaacctgt tccgcggtgt ctttgcgttt cttttatatg ttgccacctt tatgtatgta
     3361 ttttctacgt ttgctaacat actgcgtaat aaggagtctt aatgaaattt ggaaactttt
     3421 tgcttacata ccaacctccc caattttccc aaacagaggt aatgaaacgt ttggttaaat
     3481 taggtcgcat ctctgaggag tgtggttttg ataccgtatg gttactggag catcatttca
     3541 cggagtttgg tttgcttggt aacccttatg tcgctgctgc atatttactt ggcgcgacta
     3601 aaaaattgaa tgtaggaact gccgctattg ttcttcccac agcccatcca gtacgccaac
     3661 ttgaagatgt gaatttattg gatcaaatgt caaaaggacg atttcggttt ggtatttgcc
     3721 gagggcttta caacaaggac tttcgcgtat tcggcacaga tatgaataac agtcgcgcct
     3781 tagcggaatg ctggtacggg ctgataaaga atggcatgac agagggatat atggaagctg
     3841 ataatgaaca tatcaagttc cataaggtaa aagtaaaccc cgcggcgtat agcagaggtg
     3901 gcgcaccggt ttatgtggtg gctgaatcag cttcgacgac tgagtgggct gctcaatttg
     3961 gcctaccgat gatattaagt tggattataa atactaacga aaagaaagca caacttgagc
     4021 tttataatga agtggctcaa gaatatgggc acgatattca taatatcgac cattgcttat
     4081 catatataac atctgtagat catgactcaa ttaaagcgaa agagatttgc cggaaatttc
     4141 tggggcattg gtatgattct tatgtgaatg ctacgactat ttttgatgat tcagaccaaa
     4201 caagaggtta tgatttcaat aaagggcagt ggcgtgactt tgtattaaaa ggacataaag
     4261 atactaatcg ccgtattgat tacagttacg aaatcaatcc cgtgggaacg ccgcaggaat
     4321 gtattgacat aattcaaaaa gacattgatg ctacaggaat atcaaatatt tgttgtggat
     4381 ttgaagctaa tggaacagta gacgaaatta ttgcttccat gaagctcttc cagtctgatg
     4441 tcatgccatt tcttaaaaaa aaacaacgtt cgctattata ttatggcggt ggcggtagcg
     4501 gcggtggcgg tagcggcggt ggcggtagcg gcggtggcgg tagcaaattt ggattgttct
     4561 tccttaactt catcaattca acaactgttc aagaacagag tatagttcgc atgcaggaaa
     4621 taacggagta tgttgataag ttgaattttg aacagatttt agtgtatgaa aatcattttt
     4681 cagataatgg tgttgtcggc gctcctctga ctgtttctgg ttttctgctc ggtttaacag
     4741 agaaaattaa aattggttca ttaaatcaca tcattacaac tcatcatcct gtccgcatag
     4801 cggaggaagc ttgcttattg gatcagttaa gtgaagggag atttatttta gggtttagtg
     4861 attgcgaaaa aaaagatgaa atgcattttt ttaatcgccc ggttgaatat caacagcaac
     4921 tatttgaaga gtgttatgaa atcattaacg atgctttaac aacaggctat tgtaatccag
     4981 ataacgattt ttatagcttc cctaaaatat ctgtaaatcc ccatgcttat acgccaggcg
     5041 gacctcggaa atatgtaaca gcaaccagtc atcatattgt tgagtgggcg gccaaaaaag
     5101 gtattcctct catctttaag tgggatgatt ctaatgatgt tagatatgaa tatgctgaaa
     5161 gatataaagc cgttgcggat aaatatgacg ttgacctatc agagatagac catcagttaa
     5221 tgatattagt taactataac gaagatagta ataaagctaa acaagaaaca cgtgcattta
     5281 ttagtgatta tgttcttgaa atgcacccta atgaaaattt cgaaaataaa cttgaagaaa
     5341 taattgcaga aaacgctgtc ggaaattata cggagtgtat aactgcggct aagttggcaa
     5401 ttgaaaagtg tggtgcgaaa agtgtattgc tgtcctttga accaatgaat gatttgatga
     5461 gccaaaaaaa tgtaatcaat attgttgatg ataatattaa gaagtaccac acggaatata
     5521 cctaaacgga gccaatgtac gcaaaaaacc ccgcttcagc ggggtttttt cgccaaaaaa
     5581 aaccccgccc tgtcaggggc ggggtttttt tttagggccc tactgactgt aaaaaaataa
     5641 acgatgcccc ttaaagcaga agctttaagg ggcagagcgt tgcggcacat cttttcagac
     5701 ggccttattg tagcaacggt tctcatttcg tttccgaaat gagaaaggga gctacaacct
     5761 ggctctgagt agtgaggaac accacacaac atacgagccg gaagcataaa gtgtaaagcc
     5821 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     5881 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5941 ggtttgcgta ttgggcgcac ttaattaacg gcactgcgaa aaaaccccgc cgaagcgggg
     6001 ttttttgcga aatataatga ccctcttgat aacccaagag ggcatttttt aatgcccatg
     6061 gcgtttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     6121 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     6181 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     6241 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     6301 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     6361 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     6421 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     6481 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     6541 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaaaatgct
     6601 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     6661 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     6721 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     6781 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     6841 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     6901 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     6961 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     7021 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     7081 tgacattaac ctataaaaat aggcgtatca cgaggccctt gaccaaatag gc
//
