LOCUS       sequence-383225-        8271 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Base editor plasmid expressing ABE8e-NG-Cas9n under Theophylline 
            Riboswitch and constituitively expressed nonsense non-targeting 
            gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8271)
  AUTHORS   Vasquez CA, Evanoff M, Ranzau BL, Gu S, Deters E, Komor AC
  TITLE     Curing ""GFP-itis"" in Bacteria with Base Editors: Development of a 
            Genome Editing Science Program Implemented with High School Biology 
            Students.
  JOURNAL   CRISPR J. 2023 Apr 20. doi: 10.1089/crispr.2023.0002.
  PUBMED    37083425
REFERENCE   2  (bases 1 to 8271)
  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: "CRISPR J. 
            2023 Apr 20. doi: 10.1089/crispr.2023.0002."
COMMENT     Sequence Label: GFP-itis pBE-nt
FEATURES             Location/Qualifiers
     source          1..8271
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(123..914,915..983))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYYQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     746..765
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(984..1088)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1156..1174
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     CDS             complement(1196..2146)
                     /codon_start=1
                     /gene="rep101"
                     /product="RepA protein needed for replication with the 
                     pSC101 origin"
                     /label=Rep101
                     /translation="MSELVVFKANELAISRYDLTEHETKLILCCVALLNPTIENPTRKE
                     RTVSFTYNQYAQMMNISRENAYGVLAKATRELMTRTVEIRNPLVKGFEIFQWTNYAKFS
                     SEKLELVFSEEILPYLFQLKKFIKYNLEHVKSFENKYSMRIYEWLLKELTQKKTHKANI
                     EISLDEFKFMLMLENNYHEFKRLNQWVLKPISKDLNTYSNMKLVVDKRGRPTDTLIFQV
                     ELDRQMDLVTELENNQIKMNGDKIPTTITSDSYLRNGLRKTLHDALTVKIQLTSFEAKF
                     LSDMQSKHDLNGSFSWLTQKQRTTLENILAKYGRI"
     rep_origin      2194..2416
                     /label=pSC101 ori
                     /note="low-copy replication origin that requires the Rep101
                     protein"
     terminator      2981..3026
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             3791..7891
                     /codon_start=1
                     /product="Cas9 (Csn1) endonuclease from the Streptococcus 
                     pyogenes Type II CRISPR/Cas system"
                     /label=Cas9
                     /note="generates RNA-guided double strand breaks in DNA"
                     /translation="DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN
                     LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESF
                     LVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKF
                     RGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLE
                     NLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQI
                     GDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ
                     QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLR
                     KQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN
                     SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYF
                     TVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDS
                     VEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERL
                     KTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQ
                     LIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRH
                     KPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYL
                     YYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPS
                     EEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHV
                     AQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLN
                     AVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE
                     ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE
                     SIRPKRNSDKLIARKKDWDPKKYGGFVSPTVAYSVLVVAKVEKGKSKKLKSVKELLGIT
                     IMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASARFLQKGNEL
                     ALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADA
                     NLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPRAFKYFDTTIDRKVYRSTKEVLD
                     ATLIHQSITGLYETRIDLSQLGGD"
     misc_RNA        complement(8014..8089)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(join(8116..8122,8123..8140,8141..8146))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8161..8182
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tttgcgtatt gggcgcactt aattaacggc actgcgaaaa aaccccgccg aagcggggtt
       61 ttttgcgaaa tataatgacc ctcttgataa cccaagaggg cattttttaa tgcccatggc
      121 gtttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
      181 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
      241 cccagtgctg caatgatacc gcgggaccca cgctcaccgg ctccagattt atcagcaata
      301 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
      361 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
      421 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
      481 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
      541 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
      601 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
      661 tctgtgactg gtgagtactc aaccaagtca ttctgataat agtgtatgcg gcgaccgagt
      721 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
      781 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
      841 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
      901 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
      961 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     1021 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     1081 gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat tattatcatg
     1141 acattaacct ataaaaatag gcgtatcacg aggcccttag gccaaatagg ccgtttcaga
     1201 tccttccgta tttagccagt atgttctcta gtgtggttcg ttgtttttgc gtgagccatg
     1261 agaacgaacc attgagatca tgcttacttt gcatgtcact caaaaatttt gcctcaaaac
     1321 tggtgagctg aatttttaca gttaaagcat cgtgtagtgt ttttcttagt ccgttacgta
     1381 ggtaggaatc tgatgtaatg gttgttggta ttttgtcacc attcattttt atctggttgt
     1441 tctcaagttc ggttacgaga tccatttgtc tatctagttc aacttggaaa atcaacgtat
     1501 cagtcgggcg gcctcgctta tcaaccacca atttcatatt gctgtaagtg tttaaatctt
     1561 tacttattgg tttcaaaacc cattggttaa gccttttaaa ctcatggtag ttattttcaa
     1621 gcattaacat gaacttaaat tcatcaaggc taatctctat atttgccttg tgagttttct
     1681 tttgtgttag ttcttttaat aaccactcat aaatcctcat agagtatttg ttttcaaaag
     1741 acttaacatg ttccagatta tattttatga atttttttaa ctggaaaaga taaggcaata
     1801 tctcttcact aaaaactaat tctaattttt cgcttgagaa cttggcatag tttgtccact
     1861 ggaaaatctc aaagccttta accaaaggat tcctgatttc cacagttctc gtcatcagct
     1921 ctctggttgc tttagctaat acaccataag cattttccct actgatgttc atcatctgag
     1981 cgtattggtt ataagtgaac gataccgtcc gttctttcct tgtagggttt tcaatcgtgg
     2041 ggttgagtag tgccacacag cataaaatta gcttggtttc atgctccgtt aagtcatagc
     2101 gactaatcgc tagttcattt gctttgaaaa caactaattc agacatacat ctcaattggt
     2161 ctaggtgatt ttaatcacta taccaattga gatgggctag tcaatgataa ttactagtcc
     2221 ttttcctttg agttgtgggt atctgtaaat tctgctagac ctttgctgga aaacttgtaa
     2281 attctgctag accctctgta aattccgcta gacctttgtg tgtttttttt gtttatattc
     2341 aagtggttat aatttataga ataaagaaag aataaaaaaa gataaaaaga atagatccca
     2401 gccctgtgta taactcacta ctttagtcag ttccgcagta ttacaaaagg atgtcgcaaa
     2461 cgctgtttgc tcctctacaa aacagacctt aaaaccctaa aggcttaagt agcaccctcg
     2521 caagctcggg caaatcgctg aatattcctt ttgtctccga ccatcaggca cctgagtcgc
     2581 tgtctttttc gtgacattca gttcgctgcg ctcacggctc tggcagtgaa tgggggtaaa
     2641 tggcactaca ggcgcctttt atggattcat gcaaggaaac tacccataat acaagaaaag
     2701 cccgtcacgg gcttctcagg gcgttttatg gcgggtctgc tatgtggtgc tatctgactt
     2761 tttgctgttc agcagttcct gccctctgat tttccagtct gaccacttcg gattatcccg
     2821 tgacaggtca ttcagactgg ctaatgcacc cagtaaggca gcggtatcat caacaggctt
     2881 acccgtctta ctgtcctact gaaccgctct agatttcagt gcaatttatc tcttcaaatg
     2941 tagcacctga agtcagccca ggaggaagag gacatccggt caaataaaac gaaaggctca
     3001 gtcgaaagac tgggcctttc gttttagact tagggaccct cacagctaac accacgtcgt
     3061 ccctatctgc tgccctaggt ctatgagtgg ttgctggata actttacggg catgcataag
     3121 gctcgtatga tatattcagg gaagccacaa cggtttccct ctacaaataa ttttgtttaa
     3181 ctttggtgat accagcatcg tcttgatgcc cttggcagca ccctgctaag gtaacaacaa
     3241 gatgtctgag gtggagtttt cccacgagta ctggatgaga catgccctga ccctggccaa
     3301 gagggcacgg gatgagaggg aggtgcctgt gggagccgtg ctggtgctga acaatagagt
     3361 gatcggcgag ggctggaaca gagccatcgg cctgcacgac ccaacagccc atgccgaaat
     3421 tatggccctg agacagggcg gcctggtcat gcagaactac agactgattg acgccaccct
     3481 gtacgtgaca ttcgagcctt gcgtgatgtg cgccggcgcc atgatccact ctaggatcgg
     3541 ccgcgttgtg tttggcgtga ggaactcaaa aagaggcgcc gcaggctccc tgatgaacgt
     3601 gctgaactac cccggcatga atcaccgcgt cgaaattacc gagggaatcc tggcagatga
     3661 atgtgccgcc ctgctgtgcg atttctatcg gatgcctaga caggtgttca atgctcagaa
     3721 gaaggcccag agctccatca actccggtag cgaaacaccg gggacttcag aatcggccac
     3781 cccggagtct gacaagaagt acagcatcgg cctggccatc ggcaccaact ctgtgggctg
     3841 ggccgtgatc accgacgagt acaaggtgcc cagcaagaaa ttcaaggtgc tgggcaacac
     3901 cgaccggcac agcatcaaga agaacctgat cggagccctg ctgttcgaca gcggcgaaac
     3961 agccgaggcc acccggctga agagaaccgc cagaagaaga tacaccagac ggaagaaccg
     4021 gatctgctat ctgcaagaga tcttcagcaa cgagatggcc aaggtggacg acagcttctt
     4081 ccacagactg gaagagtcct tcctggtgga agaggataag aagcacgagc ggcaccccat
     4141 cttcggcaac atcgtggacg aggtggccta ccacgagaag taccccacca tctaccacct
     4201 gagaaagaaa ctggtggaca gcaccgacaa ggccgacctg cggctgatct atctggccct
     4261 ggcccacatg atcaagttcc ggggccactt cctgatcgag ggcgacctga accccgacaa
     4321 cagcgacgtg gacaagctgt tcatccagct ggtgcagacc tacaaccagc tgttcgagga
     4381 aaaccccatc aacgccagcg gcgtggacgc caaggccatc ctgtctgcca gactgagcaa
     4441 gagcagacgg ctggaaaatc tgatcgccca gctgcccggc gagaagaaga atggcctgtt
     4501 cggaaacctg attgccctga gcctgggcct gacccccaac ttcaagagca acttcgacct
     4561 ggccgaggat gccaaactgc agctgagcaa ggacacctac gacgacgacc tggacaacct
     4621 gctggcccag atcggcgacc agtacgccga cctgtttctg gccgccaaga acctgtccga
     4681 cgccatcctg ctgagcgaca tcctgagagt gaacaccgag atcaccaagg cccccctgag
     4741 cgcctctatg atcaagagat acgacgagca ccaccaggac ctgaccctgc tgaaagctct
     4801 cgtgcggcag cagctgcctg agaagtacaa agagattttc ttcgaccaga gcaagaacgg
     4861 ctacgccggc tacattgacg gcggagccag ccaggaagag ttctacaagt tcatcaagcc
     4921 catcctggaa aagatggacg gcaccgagga actgctcgtg aagctgaaca gagaggacct
     4981 gctgcggaag cagcggacct tcgacaacgg cagcatcccc caccagatcc acctgggaga
     5041 gctgcacgcc attctgcggc ggcaggaaga tttttaccca ttcctgaagg acaaccggga
     5101 aaagatcgag aagatcctga ccttccgcat cccctactac gtgggccctc tggccagggg
     5161 aaacagcaga ttcgcctgga tgaccagaaa gagcgaggaa accatcaccc cctggaactt
     5221 cgaggaagtg gtggacaagg gcgcttccgc ccagagcttc atcgagcgga tgaccaactt
     5281 cgataagaac ctgcccaacg agaaggtgct gcccaagcac agcctgctgt acgagtactt
     5341 caccgtgtat aacgagctga ccaaagtgaa atacgtgacc gagggaatga gaaagcccgc
     5401 cttcctgagc ggcgagcaga aaaaggccat cgtggacctg ctgttcaaga ccaaccggaa
     5461 agtgaccgtg aagcagctga aagaggacta cttcaagaaa atcgagtgct tcgactccgt
     5521 ggaaatctcc ggcgtggaag atcggttcaa cgcctccctg ggcacatacc acgatctgct
     5581 gaaaattatc aaggacaagg acttcctgga caatgaggaa aacgaggaca ttctggaaga
     5641 tatcgtgctg accctgacac tgtttgagga cagagagatg atcgaggaac ggctgaaaac
     5701 ctatgcccac ctgttcgacg acaaagtgat gaagcagctg aagcggcgga gatacaccgg
     5761 ctggggcagg ctgagccgga agctgatcaa cggcatccgg gacaagcagt ccggcaagac
     5821 aatcctggat ttcctgaagt ccgacggctt cgccaacaga aacttcatgc agctgatcca
     5881 cgacgacagc ctgaccttta aagaggacat ccagaaagcc caggtgtccg gccagggcga
     5941 tagcctgcac gagcacattg ccaatctggc cggcagcccc gccattaaga agggcatcct
     6001 gcagacagtg aaggtggtgg acgagctcgt gaaagtgatg ggccggcaca agcccgagaa
     6061 catcgtgatc gaaatggcca gagagaacca gaccacccag aagggacaga agaacagccg
     6121 cgagagaatg aagcggatcg aagagggcat caaagagctg ggcagccaga tcctgaaaga
     6181 acaccccgtg gaaaacaccc agctgcagaa cgagaagctg tacctgtact acctgcagaa
     6241 tgggcgggat atgtacgtgg accaggaact ggacatcaac cggctgtccg actacgatgt
     6301 ggaccatatc gtgcctcaga gctttctgaa ggacgactcc atcgacaaca aggtgctgac
     6361 cagaagcgac aagaaccggg gcaagagcga caacgtgccc tccgaagagg tcgtgaagaa
     6421 gatgaagaac tactggcggc agctgctgaa cgccaagctg attacccaga gaaagttcga
     6481 caatctgacc aaggccgaga gaggcggcct gagcgaactg gataaggccg gcttcatcaa
     6541 gagacagctg gtggaaaccc ggcagatcac aaagcacgtg gcacagatcc tggactcccg
     6601 gatgaacact aagtacgacg agaatgacaa gctgatccgg gaagtgaaag tgatcaccct
     6661 gaagtccaag ctggtgtccg atttccggaa ggatttccag ttttacaaag tgcgcgagat
     6721 caacaactac caccacgccc acgacgccta cctgaacgcc gtcgtgggaa ccgccctgat
     6781 caaaaagtac cctaagctgg aaagcgagtt cgtgtacggc gactacaagg tgtacgacgt
     6841 gcggaagatg atcgccaaga gcgagcagga aatcggcaag gctaccgcca agtacttctt
     6901 ctacagcaac atcatgaact ttttcaagac cgagattacc ctggccaacg gcgagatccg
     6961 gaagcggcct ctgatcgaga caaacggcga aaccggggag atcgtgtggg ataagggccg
     7021 ggattttgcc accgtgcgga aagtgctgag catgccccaa gtgaatatcg tgaaaaagac
     7081 cgaggtgcag acaggcggct tcagcaaaga gtctatccgt cccaagagga acagcgataa
     7141 gctgatcgcc agaaagaagg actgggaccc taagaagtac ggcggcttcg tgagccccac
     7201 cgtggcctat tctgtgctgg tggtggccaa agtggaaaag ggcaagtcca agaaactgaa
     7261 gagtgtgaaa gagctgctgg ggatcaccat catggaaaga agcagcttcg agaagaatcc
     7321 catcgacttt ctggaagcca agggctacaa agaagtgaaa aaggacctga tcatcaagct
     7381 gcctaagtac tccctgttcg agctggaaaa cggccggaag agaatgctgg cctctgcccg
     7441 gttcctgcag aagggaaacg aactggccct gccctccaaa tatgtgaact tcctgtacct
     7501 ggccagccac tatgagaagc tgaagggctc ccccgaggat aatgagcaga aacagctgtt
     7561 tgtggaacag cacaagcact acctggacga gatcatcgag cagatcagcg agttctccaa
     7621 gagagtgatc ctggccgacg ctaatctgga caaagtgctg tccgcctaca acaagcaccg
     7681 ggataagccc atcagagagc aggccgagaa tatcatccac ctgtttaccc tgaccaatct
     7741 gggagcccct agggccttca agtactttga caccaccatc gaccggaagg tgtaccgtag
     7801 caccaaagag gtgctggacg ccaccctgat ccaccagagc atcaccggcc tgtacgagac
     7861 acggatcgac ctgtctcagc tgggaggtga ctaaacggag ccaatgtacg caaaaacccc
     7921 gcttcagcgg ggttttttcg ccaaaaaaaa ccccgccctg tcaggggcgg ggtttttttt
     7981 tatgttgaaa atctccttct agattaaaaa aaagcaccga ctcggtgcca ctttttcaag
     8041 ttgataacgg actagcctta ttttaacttg ctatttctag ctctaaaact cgcatacggc
     8101 gtcgtgcact ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta
     8161 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     8221 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg g
//
