LOCUS       sequence-364088-        7853 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial PAM profiling of adenine base editor variants.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7853)
  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 7853)
  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: pTPH413
FEATURES             Location/Qualifiers
     source          1..7853
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(107..766)
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     681..700
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(767..869)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(1261..1278)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1395..1940)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(1475..1494)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        join(2052..2057,2058..2074,2075..2080)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             complement(2393..3271)
                     /codon_start=1
                     /gene="araC"
                     /product="L-arabinose regulatory protein"
                     /label=araC
                     /translation="MAEAQNDPLLPGYSFNAHLVAGLTPIEANGYLDFFIDRPLGMKGY
                     ILNLTIRGQGVVKNQGREFVCRPGDILLFPPGEIHHYGRHPEAREWYHQWVYFRPRAYW
                     HEWLNWPSIFANTGFFRPDEAHQPHFSDLFGQIINAGQGEGRYSELLAINLLEQLLLRR
                     MEAINESLHPPMDNRVREACQYISDHLADSNFDIASVAQHVCLSPSRLSHLFRQQLGIS
                     VLSWREDQRISQAKLLLSTTRMPIATVGRNVGFDDQLYFSRVFKKCTGASPSEFRAGCE
                     EKVNDVAVKLS"
     promoter        3298..3582
                     /gene="araBAD"
                     /label=araBAD promoter
                     /note="promoter of the L-arabinose operon of E. coli; the 
                     araC regulatory gene is transcribed in the opposite 
                     direction (Guzman et al., 1995)"
     primer_bind     3505..3524
                     /label=pBAD Forward
                     /note="For vectors with E. coli araBAD promoter, forward 
                     primer"
     misc_RNA        complement(7534..7626)
                     /label=Nm tracrRNA
                     /note="trans-activating CRISPR RNA for the Neisseria 
                     meningitidis CRISPR/Cas9 system (Hou et al., 2013)"
     promoter        complement(join(7682..7688,7689..7706,7707..7712))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7727..7748
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 acttaattaa cggcactgcg aaaaaacccc gccgaagcgg ggttttttgc gaaatataat
       61 gaccctcttg ataacccaag agggcatttt ttaatgccca tggcgtttac gccccgccct
      121 gccactcatc gcagtactgt tgtaattcat taagcattct gccgacatgg aagccatcac
      181 aaacggcatg atgaacctga atcgccagcg gcatcagcac cttgtcgcct tgcgtataat
      241 atttgcccat ggtgaaaacg ggggcgaaga agttgtccat attggccacg tttaaatcaa
      301 aactggtgaa actcacccag ggattggctg agacgaaaaa catattctca ataaaccctt
      361 tagggaaata ggccaggttt tcaccgtaac acgccacatc ttgcgaatat atgtgtagaa
      421 actgccggaa atcgtcgtgg tattcactcc agagcgatga aaacgtttca gtttgctcat
      481 ggaaaacggt gtaacaaggg tgaacactat cccatatcac cagctcaccg tctttcattg
      541 ccatacggaa ttccggatga gcattcatca ggcgggcaag aatgtgaata aaggccggat
      601 aaaacttgtg cttatttttc tttacggtct ttaaaaaggc cgtaatatcc agctgaacgg
      661 tctggttata ggtacattga gcaactgact gaaatgcctc aaaatgttct ttacgatgcc
      721 attgggatat atcaacggtg gtatatccag tgattttttt ctccatttta gcttccttag
      781 ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag tgatcttatt tcattatggt
      841 gaaagttgga acctcttacg tgccgatcaa cgtctcattt tcgccaaaag ttggcccggg
      901 gcttcccggt atcaacaggg acaccaggat ttatttattc tgcgaagtga tcttccgtca
      961 caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc caacttactg atttagtgta
     1021 tgatggtgtt tttgaggtgc tccagtggct tctgtttcta tcagctgtcc ctcctgttca
     1081 gctactgacg gggtggtgcg taacggcaaa agcaccgccg gacatcagcg ctagcggagt
     1141 gtatactggc ttactatgtt ggcactgatg agggtgtcag tgaagtgctt catgtggcag
     1201 gagaaaaaag gctgcaccgg tgcgtcagca gaatatgtga tacaggatat attccgcttc
     1261 ctcgctcact gactcgctac gctcggtcgt tcgactgcgg cgagcggaaa tggcttacga
     1321 acggggtgga gatttcctgg aagatgccag gaagatactt aacagggaag tgagagggcc
     1381 gcggcaaagc cgtttttcca taggctccgc ccccctgaca agcatcacga aatctgacgc
     1441 tcaaatcagt ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctggc
     1501 ggctccctcg tgcgctctcc tgttcctgcc tttcggttta ccggtgtcat tccgctgtta
     1561 tggccgcgtt tgtctcattc cacgcctgac actcagttcc gggtaggcag ttcgctccaa
     1621 gctggactgt atgcacgaac cccccgttca gtccgaccgc tgcgccttat ccggtaacta
     1681 tcgtcttgag tccaacccgg aaaaacatgc aaaagcacca ctggcagcag ccactggtaa
     1741 ttgatttaga ggagttagtc ttgaagtcat gcgccggtta aggctaaact gaaaggacaa
     1801 gttttggtga ctgcgctcct ccaagccagt tacctcggtt caaagagttg gtagctcaga
     1861 gaaccttcga aaaaccgccc tgcaaggcgg ttttttcgtt ttcagagcaa gagattacgc
     1921 gcagaccaaa acgatctcaa gaagatcatc ttattaatca gataaaatat ttctagattt
     1981 cagtgcaatt tatctcttca aatgtagcac ctgaagtcag ccccatacga tataagttgt
     2041 aattctaatg tttgacagct tatcatcgat aagctttaat gcggtagttt atcacagtta
     2101 aattgctaac gcagtcaggc accgtgtatg aaatctaaca atgcgctcat cgtcatcctc
     2161 ggcaccgtca ccctggatgc tgtaggcata ggcttggtta tgccggtact gccgggcctc
     2221 ttgcgggaaa gaggacatcc ggtcaaataa aacgaaaggc tcagtcgaaa gactgggcct
     2281 ttcgttttgc tgaggagact takggrssnw wwnaggacat ccggtcaaat aaaacgaaag
     2341 gctcagtcga aagactgggc ctttcgtttt gctgaggaga cttagggacc ctttatgaca
     2401 acttgacggc tacatcattc actttttctt cacaaccggc acggaactcg ctcgggctgg
     2461 ccccggtgca ttttttaaat acccgcgaga aatagagttg atcgtcaaaa ccaacattgc
     2521 gaccgacggt ggcgataggc atccgggtgg tgctcaaaag cagcttcgcc tggctgatac
     2581 gttggtcctc gcgccagctt aagacgctaa tccctaactg ctggcggaaa agatgtgaca
     2641 gacgcgacgg cgacaagcaa acatgctgtg cgacgctggc gatatcaaaa ttgctgtctg
     2701 ccaggtgatc gctgatgtac tgacaagcct cgcgtacccg attatccatc ggtggatgga
     2761 gcgactcgtt aatcgcttcc atgcgccgca gtaacaattg ctcaagcaga tttatcgcca
     2821 gcagctccga atagcgccct tccccttgcc cggcgttaat gatttgccca aacaggtcgc
     2881 tgaaatgcgg ctggtgcgct tcatccgggc gaaagaaccc cgtattggca aatattgacg
     2941 gccagttaag ccattcatgc cagtaggcgc gcggacgaaa gtaaacccac tggtgatacc
     3001 attcgcgagc ctccggatga cgaccgtagt gatgaatctc tcctggcggg aacagcaaaa
     3061 tatcacccgg tcggcaaaca aattctcgtc cctgattttt caccaccccc tgaccgcgaa
     3121 tggtgagatt gagaatataa cctttcattc ccagcggtcg gtcgataaaa aaatcgagat
     3181 aaccgttggc ctcaatcggc gttaaacccg ccaccagatg ggcattaaac gagtatcccg
     3241 gcagcagggg atcattttgc gcttcagcca tacttttcat actcccacca ttcagagaag
     3301 aaaccaattg tccatattgc atcagacatt gccgtcactg cgtcttttac tggctcttct
     3361 cgctaaccca accggtaacc ccgcttatta aaagcattct gtaacaaagc gggaccaaag
     3421 ccatgacaaa aacgcgtaac aaaagtgtct ataatcacgg cagaaaagtc cacattgatt
     3481 atttgcacgg cgtcacactt tgctatgcca tagcattttt atccataaga ttagcggatc
     3541 ctacctgacg ctttttatcg caactctcta ctgtttctcc atacccgttt ttttacctgc
     3601 aggtgcagta aaaaaaggaa aaattgtctg aggtggagtt ttcccacgag tactggatga
     3661 gacatgccct gaccctggcc aagagggcac gggatgagag ggaggtgcct gtgggagccg
     3721 tgctggtgct gaacaataga gtgatcggcg agggctggaa cagagccatc ggcctgcacg
     3781 acccaacagc ccatgccgaa attatggccc tgagacaggg cggcctggtc atgcagaact
     3841 acagactgat tgacgccacc ctgtacgtga cattcgagcc ttgcgtgatg tgcgccggcg
     3901 ccatgatcca ctctaggatc ggccgcgtgg tgtttggcgt gaggaactca aaaagaggcg
     3961 ccgcaggctc cctgatgaac gtgctgaact accccggcat gaatcaccgc gtcgaaatta
     4021 ccgagggaat cctggcagat gaatgtgccg ccctgctgtg cgatttctat cggatgccta
     4081 gacaggtgtt caatgctcag aagaaggccc agagctccat caacaaggaa tctggctctg
     4141 tatcttctga acagttggcg caattccgct ctctggacgc agcattcaag cctaacccaa
     4201 tcaattacat actgggactg gcaatcggaa tcgcatccgt gggatgggct atggtggaga
     4261 tcgacgagga ggggaatcct atccggctga tcgatctggg cgtgagagtg tttgagaggg
     4321 ccgaggtgcc aaagaccggc gattctctgg ctatggcccg gagactggca cggagcgtga
     4381 ggcgcctgac acggagaagg gcacacaggc tgctgagggc acgccggctg ctgaagagag
     4441 agggcgtgct gcaggcagca gacttcgatg agaatggcct gatcacgagc ttgccaaaca
     4501 ccccctggca gctgagagca gccgccctgg acaggaagct gacaccactg gagtggtctg
     4561 ccgtgctgct gcacctgatc aagcaccgcg gctacctgag ccagcggaag aacgagggag
     4621 agacagcagc caaggagctg ggcgccctgc tgaagggagt ggccaacaat gcccacgccc
     4681 tgcagaccgg cgatttcagg acacctgccg agctggccct gaataagttt gagaaggagt
     4741 ccggccacat cagaaaccag aggggcgact atagccacac cttctcccgc aaggatctgc
     4801 aggccgagct gatcctgctg ttcgagaagc agaaggagtt tggcaatcca cacgtgagcg
     4861 gaggcctgaa ggagggaatc gagaccctgc tgatgacaca gaggcctgcc ctgtccggcg
     4921 acgcagtgca gaagatgctg gggcactgca ccctcgagcc tacagagcca aaggccgcca
     4981 agaacaccta cacagccgag cggtttatct ggctgacaaa gctgaacaat ctgagaatcc
     5041 tggagcaggg atccgagagg ccactgaccg acacagagag gtccaccctg atggatgagc
     5101 cttaccggaa gtctaaactg acatatgccc aggccagaaa gctgctgggc ctggaggaca
     5161 ccgccttctt taagggcctg agatacggca aggataatgc cgaggcctcc acactgatgg
     5221 agatgaaggc ctatcacgcc atctctcgcg ccctggagaa ggagggcctg aaggacaaga
     5281 agtcccccct gaacctgagc tccgagctgc aggatgagat cggcaccgcc ttctctctgt
     5341 ttaagaccga cgaggatatc acaggccgcc tgaaggacag ggtgcagcct gagatcctgg
     5401 aggccctgct gaagcacatc tctttcgata agtttgtgca gatcagcctg aaggccctga
     5461 gaaggatcgt gccactgatg gagcagggca agcggtacga cgaggcctgc gccgagatct
     5521 acggcgttca ctatggcaag aagaacacag aggagaagat ctatctgccc cctatccctg
     5581 ccgacgagat cagaaatcct gtggtgctga gggccctgtc ccaggcaaga aaagtgatca
     5641 acggagtggt gcgccggtac ggatctccag cccggatcca catcgagacc gccagagaag
     5701 tgggcaagag cttcaaggac cggaaggaga tcgcgaagag acaggaggag aatcgcaagg
     5761 atcgggagaa ggccgccgcc aagtttaggg agtacttccc taactttgtg ggcgagccaa
     5821 agtctaagga catcctgaag ctgcgcctgt acgagcagca gcacggcaag tgtctgtata
     5881 gcggcaaaga gatcaatctg gtgcggctga acgagaaggg ctatgtggag atcgatgcag
     5941 ccctgccttt ctccagaacc tgggacgatt cttttaacaa taaggtgctg gtgctgggca
     6001 gcgagaacca gaataagggc aatcagacac catacgagta tttcaatggc aaggacaact
     6061 ccagggagtg gcaggagttc aaggcccgcg tggagacctc tagatttccc agtagcaaga
     6121 agcagcggat cctgctgcag aagttcgacg aggatggctt taaggagtgc aacctgaatg
     6181 acaccagata cgtgaaccgg ttcctgtgcc agtttgtggc cgatcacatc ctgctgaccg
     6241 gcaagggcaa gagaagggtg gtcgcctcta atggccagat cacaaacctg ctgagggggt
     6301 tttggagact gaggaaggtg cgggcagaga atgacagaca ccacgcactg gatgcagtgg
     6361 tggtggcatg cagcaccgtg gcaatgcagc agaagatcac aagattcgtg aggtataagg
     6421 agatgaacgc ctttgacggc aagaccgtcg ataaggagac aggcaaggtg ctgtaccaga
     6481 agacccactt cccccagcct tgggagttct ttgcccagga agttatgatc cgggtgttcg
     6541 gcaagccaga cggcaagcct gagtttgagg aggccgatac cccagagaag ctgaggacac
     6601 tgctggcaga gaagctgtct agcaggccag aggcagtgca cgagtacgtg accccgctgt
     6661 tcgtgtccag ggcacccaat cggaagatgt ctggcgccca caaggacaca ctgagaagcg
     6721 ccaagaggtt tgtgaagcac aacgagaaga tctccgtgaa gagagtgtgg ctgaccgaga
     6781 tcaagctggc cgatctggag aacatggtga attacaagaa cggcagggag atcgagctgt
     6841 atgaggccct gaaggcaagg ctggaggcct acggaggaaa tgccaagcag gccttcgacc
     6901 caaaggataa ccccttttat aagaagggag gacagctggt gaaggccgtg cgggtggaga
     6961 agacccagaa gagcggcgtg ctgctgaata agaagaacgc ctacacaatc gccgacaatg
     7021 gtgatatggt gagagtggac gtgttctgta aggtggataa gaagggcaag aatcagtact
     7081 ttatcgtgcc tatctatgcc tggcaggtgg ccgagaacat cctgccagac atcgattgca
     7141 agggctacag aatcgacgat agctatacat tctgtttttc cctgcacaag tatgacctga
     7201 tcgccttcca gaaggatgag aagtccaagg tggagtttgc ctactatatc aattgcgact
     7261 cctctagcgg cgggttctac ctggcctggc acgataaggg cagcagggag cagcggtttc
     7321 gcatctccac ccagaatctg gcgctgatcc agaagtatca ggtgaacgag ctgggcaagg
     7381 agatcaggcc atgtcggctg aagaagcgcc cacccgtgta aacggagcca atgtacgcaa
     7441 aaaacctcgc ttcagcgggg ttttttcgcc aaaaaaaacc ccgccctgtc aggggcgggg
     7501 ttttttttta gggccctact gactgtaaaa aaataaacga tgccccttaa agcagaagct
     7561 ttaaggggca gagcgttgcg gcacatcttt tcagacggcc ttattgtagc aacggttctc
     7621 atttcgtttc cgaaatgaga aagggagcta caactcacgt gctcagtctg ggccatccac
     7681 acaacatacg agccggaaac ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     7741 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     7801 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgc
//
