LOCUS       sequence-365708-        7171 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 7171)
  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 7171)
  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: pTPH418b
FEATURES             Location/Qualifiers
     source          1..7171
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     52..70
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     rep_origin      89..677
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     578..597
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     831..848
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    863..1003
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(929..948)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1089..1111
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(1105..1296)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     terminator      1407..1453
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             3527..4606
                     /codon_start=1
                     /gene="luxA"
                     /product="LuxA luciferase subunit"
                     /label=LuxA
                     /note="from the Photorhabdus luminescens bioluminescence 
                     operon"
                     /translation="MKFGNFLLTYQPPQFSQTEVMKRLVKLGRISEECGFDTVWLLEHH
                     FTEFGLLGNPYVAAAYLLGATKKLNVGTAAIVLPTAHPVRQLEDVNLLDQMSKGRFRFG
                     ICRGLYNKDFRVFGTDMNNSRALAECWYGLIKNGMTEGYMEADNEHIKFHKVKVNPAAY
                     SRSGAPVYVVAESASTTEWAAQFGLPMILSWIINTNEKKAQLELYNEVAQEYGHDIHNI
                     DHCLSYITSVDHDSIKAKEICRKFLGHWYDSYVNATTIFDDSDQTRGYDFNKGQWRDFV
                     LKGHKDTNRRIDYSYEINPVGTPQECIDIIQKDIDATGISNICCGFEANGTVDEIIASM
                     KLFQSDVMPFLKKKQRSLLY"
     CDS             4670..5650
                     /codon_start=1
                     /gene="luxB"
                     /product="LuxB luciferase subunit"
                     /label=LuxB
                     /note="from the Photorhabdus luminescens bioluminescence 
                     operon"
                     /translation="KFGLFFLNFINSTTVQEQSIVRMQEITEYVDKLNFEQILVYENHF
                     SDNGVVGAPLTVSGFLLGLTEKIKIGSLNHIIITHHPVRIAEEACLLDQLSEGRFILGF
                     SDCEKKDEMHFFNRPVEYQQQLFEECYEIINDALTTGYCNPDNDFYSFPKISVNPHAYT
                     PGGPRKYVTATSHHIVEWAAKKGIPLIFKWDDSNDVRYEYAERYKAVADKYDVDLSEID
                     HQLMILVNYNEDSNKAKQETRAFISDYVLEMHPNENFENKLEEIIAENAVGNYTECITA
                     AKLAIEKCGAKSVLLSFEPMNDLMSQKNVINIVDDNIKKYHTEYT"
     misc_RNA        complement(5763..5855)
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     promoter        complement(join(5912..5918,5919..5936,5937..5942))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5957..5978
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(join(6190..6981,6982..7050))
                     /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     6813..6832
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7051..7155)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt
       61 atcacgaggc ccttgaccaa ataggccgtt gagatccttt ttttctgcgc gtaatctgct
      121 gcttgtaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
      181 cacctctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc
      241 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
      301 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
      361 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
      421 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
      481 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
      541 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
      601 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
      661 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
      721 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
      781 cagcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
      841 tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat ctgtgcggta
      901 tttcacaccg catatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
      961 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     1021 acccgctgac gcgccctgac gggcttgtct tctcccggca tccgcttaca gacaagctgt
     1081 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     1141 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     1201 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     1261 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     1321 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     1381 tgatgatgaa caagaggaca tccggtcaaa taaaacgaaa ggctcagtcg aaagactggg
     1441 cctttcgttt tatggaaaga agagaaaccg ggactggttc gttttgctga ggagacttag
     1501 ggactctcac agctaacacc acgtcgtccc tatctgctgc cctaggtcta tgagtggttg
     1561 ctggataact ttacgggcat gcataaggct cgtatgatat attcagggag accacaacgg
     1621 tttccctcta caaataattt tgtttaactt ttactagagt gggaccctac ctgcaggtgc
     1681 agtaaaggaa aaaaaaaatg aaaaaattat tatttgcgat accgctgtgc ttaagctacg
     1741 aaactgaaat cttgaccgtc gaatatggtc tgctgccaat cggcaagatt gttgaaaaac
     1801 gtattgaatg tacggtctac tcagtggata acaacggcaa tatctacacc cagccggtgg
     1861 cccagtggca tgaccgtggt gaacaggaag tgttcgaata ttgtctggaa gacggatctt
     1921 taatccgtgc cacaaaggat cacaaattta tgactgtaga tggtcagatg ctcccaatcg
     1981 acgaaatttt tgaacgcgaa ttagacctga tgcgcgtgga taatctcccg aattccggag
     2041 gatctagccc aacctctggc tctgagtagg gcggaacagg atctagcggg ggttcaagcg
     2101 agagtgcaac acctgaaagc agcgggacaa aagctggctc tgagtagggc ggaacaagcg
     2161 gtggctcaat caaaattgcc acgcgtaaat atttaggcaa acagaatgtt tatgatatcg
     2221 gtgtcgagcg cgatcataat ttcgcgctga aaaacggatt tatagctagc aattgtttca
     2281 acgttgttcc tttctattct cactccgccg aaactgttga aagttgttta gcaaaacccc
     2341 atacagaaaa ttcatttact aacgtctgga aagacgacaa aactttagat cgttacgcta
     2401 actatgaggg ctgtctgtgg aatgctacag gcgttgtagt ttgtactggt gacgaaactc
     2461 agtgttacgg tacatgggtt cctattgggc ttgctatccc tgaaaatgag ggtggtggct
     2521 ctgagggtgg cggttctgag ggtggcggtt ctgagggtgg cggtactaaa cctcctgagt
     2581 acggtgatac acctattccg ggctatactt atatcaaccc tctcgacggc acttatccgc
     2641 ctggtactga gcaaaacccc gctaatccta atccttctct tgaggagtct cagcctctta
     2701 atactttcat gtttcagaat aataggttcc gaaataggca gggggcatta actgtttata
     2761 cgggcactgt tactcaaggc actgaccccg ttaaaactta ttaccagtac actcctgtat
     2821 catcaaaagc catgtatgac gcttactgga acggtaaatt cagagactgc gctttccatt
     2881 ctggctttaa tgaggatcca ttcgtttgtg aatatcaagg ccaatcgtct gacctgcctc
     2941 aacctcctgt caatgctggc ggcggctctg gtggtggttc tggtggcggc tctgagggtg
     3001 gtggctctga gggtggcggt tctgagggtg gcggctctga gggaggcggt tccggtggtg
     3061 gctctggttc cggtgatttt gattatgaaa agatggcaaa cgctaataag ggggctatga
     3121 ccgaaaatgc cgatgaaaac gcgctacagt ctgacgctaa aggcaaactt gattctgtcg
     3181 ctactgatta cggtgctgct atcgatggtt tcattggtga cgtttccggc cttgctaatg
     3241 gtaatggtgc tactggtgat tttgctggct ctaattccca aatggctcaa gtcggtgacg
     3301 gtgataattc acctttaatg aataatttcc gtcaatattt accttccctc cctcaatcgg
     3361 ttgaatgtcg cccttttgtc tttggcgctg gtaaacctta cgagttcagt atcgactgcg
     3421 ataagatcaa cctgttccgc ggtgtctttg cgtttctttt atatgttgcc acctttatgt
     3481 atgtattttc tacgtttgct aacatactgc gtaataagga gtcttaatga aatttggaaa
     3541 ctttttgctt acataccaac ctccccaatt ttcccaaaca gaggtaatga aacgtttggt
     3601 taaattaggt cgcatctctg aggagtgtgg ttttgatacc gtatggttac tggagcatca
     3661 tttcacggag tttggtttgc ttggtaaccc ttatgtcgct gctgcatatt tacttggcgc
     3721 gactaaaaaa ttgaatgtag gaactgccgc tattgttctt cccacagccc atccagtacg
     3781 ccaacttgaa gatgtgaatt tattggatca aatgtcaaaa ggacgatttc ggtttggtat
     3841 ttgccgaggg ctttacaaca aggactttcg cgtattcggc acagatatga ataacagtcg
     3901 cgccttagcg gaatgctggt acgggctgat aaagaatggc atgacagagg gatatatgga
     3961 agctgataat gaacatatca agttccataa ggtaaaagta aaccccgcgg cgtatagcag
     4021 aagtggcgca ccggtttatg tggtggctga atcagcttcg acgactgagt gggctgctca
     4081 atttggccta ccgatgatat taagttggat tataaatact aacgaaaaga aagcacaact
     4141 tgagctttat aatgaagtgg ctcaagaata tgggcacgat attcataata tcgaccattg
     4201 cttatcatat ataacatctg tagatcatga ctcaattaaa gcgaaagaga tttgccggaa
     4261 atttctgggg cattggtatg attcttatgt gaatgctacg actatttttg atgattcaga
     4321 ccaaacaaga ggttatgatt tcaataaagg gcagtggcgt gactttgtat taaaaggaca
     4381 taaagatact aatcgccgta ttgattacag ttacgaaatc aatcccgtgg gaacgccgca
     4441 ggaatgtatt gacataattc aaaaagacat tgatgctaca ggaatatcaa atatttgttg
     4501 tggatttgaa gctaatggaa cagtagacga aattattgct tccatgaagc tcttccagtc
     4561 tgatgtcatg ccatttctta aaaaaaaaca acgttcgcta ttatattatg gcggtggcgg
     4621 tagcggcggt ggcggtagcg gcggtggcgg tagcggcggt ggcggtagca aatttggatt
     4681 gttcttcctt aacttcatca attcaacaac tgttcaagaa cagagtatag ttcgcatgca
     4741 ggaaataacg gagtatgttg ataagttgaa ttttgaacag attttagtgt atgaaaatca
     4801 tttttcagat aatggtgttg tcggcgctcc tctgactgtt tctggttttc tgctcggttt
     4861 aacagagaaa attaaaattg gttcattaaa tcacatcatt ataactcatc atcctgtccg
     4921 catagcggag gaagcttgct tattggatca gttaagtgaa gggagattta ttttagggtt
     4981 tagtgattgc gaaaaaaaag atgaaatgca tttttttaat cgcccggttg aatatcaaca
     5041 gcaactattt gaagagtgtt atgaaatcat taacgatgct ttaacaacag gctattgtaa
     5101 tccagataac gatttttata gcttccctaa aatatctgta aatccccatg cttatacgcc
     5161 aggcggacct cggaaatatg taacagcaac cagtcatcat attgttgagt gggcggccaa
     5221 aaaaggtatt cctctcatct ttaagtggga tgattctaat gatgttagat atgaatatgc
     5281 tgaaagatat aaagccgttg cggataaata tgacgttgac ctatcagaga tagaccatca
     5341 gttaatgata ttagttaact ataacgaaga tagtaataaa gctaaacaag aaacacgtgc
     5401 atttattagt gattatgttc ttgaaatgca ccctaatgaa aatttcgaaa ataaacttga
     5461 agaaataatt gcagaaaacg ctgtcggaaa ttatacggag tgtataactg cggctaagtt
     5521 ggcaattgaa aagtgtggtg cgaaaagtgt attgctgtcc tttgaaccaa tgaatgattt
     5581 gatgagccaa aaaaatgtaa tcaatattgt tgatgataat attaagaagt accacacgga
     5641 atatacctaa acggagccaa tgtacgcaaa aaaccccgct tcagcggggt tttttcgcca
     5701 aaaaaaaccc cgccctgtca ggggcggggt ttttttttag ggccctactg actgtaaaaa
     5761 aataaacgat gccccttaaa gcagaagctt taaggggcag agcgttgcgg cacatctttt
     5821 cagacggcct tattgtagca acggttctca tttcgtttcc gaaatgagaa agggagctac
     5881 aacctggctc tgagtagggc ggaacaccac acaacatacg agccggaagc ataaagtgta
     5941 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
     6001 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
     6061 gaggcggttt gcgtattggg cgcacttaat taacggcact gcgaaaaaac cccgccgaag
     6121 cggggttttt tgcgaaatat aatgaccctc ttgataaccc aagagggcat tttttaatgc
     6181 ccatggcgtt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6241 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6301 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6361 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6421 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6481 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6541 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6601 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6661 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaaa
     6721 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6781 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6841 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6901 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6961 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7021 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7081 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7141 aataggggtt ccgcgcacat ttccccgaaa a
//
