LOCUS       CGBE1_(pRZ3885).        9909 bp DNA     circular SYN 13-MAY-2021
DEFINITION  CMV promoter expression plasmid for 
            UNG(E.coli)-rAPOBEC1(R33A)-nCas9-P2A-EGFP.
ACCESSION   .
VERSION     .
KEYWORDS    CGBE1 (pRZ3885).
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9909)
  AUTHORS   Kurt IC, Zhou R, Iyer S, Garcia SP, Miller BR, Langner LM, Grunewald
            J, Joung JK
  TITLE     CRISPR C-to-G base editors for inducing targeted DNA transversions 
            in human cells.
  JOURNAL   Nat Biotechnol. 2020 Jul 20. pii: 10.1038/s41587-020-0609-x. doi: 
            10.1038/s41587-020-0609-x.
  PUBMED    32690971
REFERENCE   2  (bases 1 to 9909)
  TITLE     Direct Submission
REFERENCE   3  (bases 1 to 9909)
  AUTHORS   .
  TITLE     Direct Submission
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Biotechnol. 2020 Jul 20. pii: 10.1038/s41587-020-0609-x. doi: 
            10.1038/s41587-020-0609-x."
COMMENT     SGRef: number: 2; type: "Journal Article"
FEATURES             Location/Qualifiers
     source          1..9909
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     220..239
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     473..490
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    607..628
                     /label=CAP binding site
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
     promoter        643..673
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    681..697
                     /label=lac operator
                     /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)."
     primer_bind     705..721
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    complement(792..1016)
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     CDS             complement(1045..1062)
                     /label=6xHis
                     /note="6xHis affinity tag"
     CDS             complement(1071..1787)
                     /label=EGFP
                     /note="enhanced GFP"
     CDS             complement(1788..1844)
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             complement(1863..1883)
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     CDS             complement(1926..6026)
                     /label=Cas9(D10A)
                     /note="nickase mutant of the Cas9 endonuclease from the 
                     Streptococcus pyogenes Type II CRISPR/Cas system"
     CDS             complement(6123..6806)
                     /label=APOBEC-1
                     /note="cytidine deaminase (C to U editing enzyme) from rat"
     CDS             complement(6837..7523)
                     /gene="ung"
                     /label=ung
                     /note="Uracil-DNA glycosylase from Escherichia coli (strain
                     K12). Accession#: P12295"
     CDS             complement(7521..7541)
                     /label=SV40 NLS
                     /note="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
     promoter        complement(7591..7609)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(7651..7854)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
     enhancer        complement(7855..8234)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     8406..8425
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
     promoter        8504..8608
                     /label=AmpR promoter
     CDS             8609..9466
                     /label=AmpR
                     /note="beta-lactamase"
     rep_origin      9640..9909
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
       61 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
      121 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
      181 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta
      241 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
      301 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
      361 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
      421 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
      481 agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg cgcgttggcc
      541 gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa
      601 cgcaattaat gtgagttagc tcactcatta ggcaccctag gctttacact ttatgcttcc
      661 ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga
      721 ccatgattac gccaagctct agctagaggt cgacggtatc gagccccagc tggttctttc
      781 cgcctcagaa gccatagagc ccaccgcatc cccagcatgc ctgctattgt cttcccaatc
      841 ctcccccttg ctgtcctgcc ccaccccacc ccccagaata gaatgacacc tactcagaca
      901 atgcgatgca atttcctcat tttattagga aaggacagtg ggagtggcac cttccagggt
      961 caaggaaggc acgggggagg ggcaaacaac agatggctgg caactagaag gcacagtcga
     1021 ggctgatcag cgggtttaaa ctcaatggtg atggtgatga tgaccggtca cttgtacagc
     1081 tcgtccatgc cgagagtgat cccggcggcg gtcacgaact ccagcaggac catgtgatcg
     1141 cgcttctcgt tggggtcttt gctcagggcg gactgggtgc tcaggtagtg gttgtcgggc
     1201 agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc gagctgcacg
     1261 ctgccgtcct cgatgttgtg gcggatcttg aagttcacct tgatgccgtt cttctgcttg
     1321 tcggccatga tatagacgtt gtggctgttg tagttgtact ccagcttgtg ccccaggatg
     1381 ttgccgtcct ccttgaagtc gatgcccttc agctcgatgc ggttcaccag ggtgtcgccc
     1441 tcgaacttca cctcggcgcg ggtcttgtag ttgccgtcgt ccttgaagaa gatggtgcgc
     1501 tcctggacgt agccttcggg catggcggac ttgaagaagt cgtgctgctt catgtggtcg
     1561 gggtagcggc tgaagcactg cacgccgtag gtcagggtgg tcacgagggt gggccagggc
     1621 acgggcagct tgccggtggt gcagatgaac ttcagggtca gcttgccgta ggtggcatcg
     1681 ccctcgccct cgccggacac gctgaacttg tggccgttta cgtcgccgtc cagctcgacc
     1741 aggatgggca ccaccccggt gaacagctcc tcgcccttgc tcaccatcgg tccaggattc
     1801 tcttcgacat ctccggcttg tttcagcaga gagaagtttg ttgcgccgct tccaccgcct
     1861 ccgactttcc tcttcttctt gggctcgaat tcgctgccgt cggcggttct ttttgagccg
     1921 ccagagtcac ctcccagctg agacaggtcg atccgtgtct cgtacaggcc ggtgatgctc
     1981 tggtggatca gggtggcgtc cagcacctct ttggtgctgg tgtacctctt ccggtcgatg
     2041 gtggtgtcaa agtacttgaa ggcggcaggg gctcccagat tggtcagggt aaacaggtgg
     2101 atgatattct cggcctgctc tctgatgggc ttatcccggt gcttgttgta ggcggacagc
     2161 actttgtcca gattagcgtc ggccaggatc actctcttgg agaactcgct gatctgctcg
     2221 atgatctcgt ccaggtagtg cttgtgctgt tccacaaaca gctgtttctg ctcattatcc
     2281 tcgggggagc ccttcagctt ctcatagtgg ctggccaggt acaggaagtt cacatatttg
     2341 gagggcaggg ccagttcgtt tcccttctgc agttcgccgg cagaggccag cattctcttc
     2401 cggccgtttt ccagctcgaa cagggagtac ttaggcagct tgatgatcag gtcctttttc
     2461 acttctttgt agcccttggc ttccagaaag tcgatgggat tcttctcgaa gctgcttctt
     2521 tccatgatgg tgatccccag cagctctttc acactcttca gtttcttgga cttgcccttt
     2581 tccactttgg ccaccaccag cacagaatag gccacggtgg ggctgtcgaa gccgccgtac
     2641 ttcttagggt cccagtcctt ctttctggcg atcagcttat cgctgttcct cttgggcagg
     2701 atagactctt tgctgaagcc gcctgtctgc acctcggtct ttttcacgat attcacttgg
     2761 ggcatgctca gcactttccg cacggtggca aaatcccggc ccttatccca cacgatctcc
     2821 ccggtttcgc cgtttgtctc gatcagaggc cgcttccgga tctcgccgtt ggccagggta
     2881 atctcggtct tgaaaaagtt catgatgttg ctgtagaaga agtacttggc ggtagccttg
     2941 ccgatttcct gctcgctctt ggcgatcatc ttccgcacgt cgtacacctt gtagtcgccg
     3001 tacacgaact cgctttccag cttagggtac tttttgatca gggcggttcc cacgacggcg
     3061 ttcaggtagg cgtcgtgggc gtggtggtag ttgttgatct cgcgcacttt gtaaaactgg
     3121 aaatccttcc ggaaatcgga caccagcttg gacttcaggg tgatcacttt cacttcccgg
     3181 atcagcttgt cattctcgtc gtacttagtg ttcatccggg agtccaggat ctgtgccacg
     3241 tgctttgtga tctgccgggt ttccaccagc tgtctcttga tgaagccggc cttatccagt
     3301 tcgctcaggc cgcctctctc ggccttggtc agattgtcga actttctctg ggtaatcagc
     3361 ttggcgttca gcagctgccg ccagtagttc ttcatcttct tcacgacctc ttcggagggc
     3421 acgttgtcgc tcttgccccg gttcttgtcg cttctggtca gcaccttgtt gtcgatggag
     3481 tcgtccttca gaaagctctg aggcacgata tggtccacat cgtagtcgga cagccggttg
     3541 atgtccagtt cctggtccac gtacatatcc cgcccattct gcaggtagta caggtacagc
     3601 ttctcgttct gcagctgggt gttttccacg gggtgttctt tcaggatctg gctgcccagc
     3661 tctttgatgc cctcttcgat ccgcttcatt ctctcgcggc tgttcttctg tcccttctgg
     3721 gtggtctggt tctctctggc catttcgatc acgatgttct cgggcttgtg ccggcccatc
     3781 actttcacga gctcgtccac caccttcact gtctgcagga tgcccttctt aatggcgggg
     3841 ctgccggcca gattggcaat gtgctcgtgc aggctatcgc cctggccgga cacctgggct
     3901 ttctggatgt cctctttaaa ggtcaggctg tcgtcgtgga tcagctgcat gaagtttctg
     3961 ttggcgaagc cgtcggactt caggaaatcc aggattgtct tgccggactg cttgtcccgg
     4021 atgccgttga tcagcttccg gctcagcctg ccccagccgg tgtatctccg ccgcttcagc
     4081 tgcttcatca ctttgtcgtc gaacaggtgg gcataggttt tcagccgttc ctcgatcatc
     4141 tctctgtcct caaacagtgt cagggtcagc acgatatctt ccagaatgtc ctcgttttcc
     4201 tcattgtcca ggaagtcctt gtccttgata attttcagca gatcgtggta tgtgcccagg
     4261 gaggcgttga accgatcttc cacgccggag atttccacgg agtcgaagca ctcgattttc
     4321 ttgaagtagt cctctttcag ctgcttcacg gtcactttcc ggttggtctt gaacagcagg
     4381 tccacgatgg cctttttctg ctcgccgctc aggaaggcgg gctttctcat tccctcggtc
     4441 acgtatttca ctttggtcag ctcgttatac acggtgaagt actcgtacag caggctgtgc
     4501 ttgggcagca ccttctcgtt gggcaggttc ttatcgaagt tggtcatccg ctcgatgaag
     4561 ctctgggcgg aagcgccctt gtccaccact tcctcgaagt tccagggggt gatggtttcc
     4621 tcgctctttc tggtcatcca ggcgaatctg ctgtttcccc tggccagagg gcccacgtag
     4681 taggggatgc ggaaggtcag gatcttctcg atcttttccc ggttgtcctt caggaatggg
     4741 taaaaatctt cctgccgccg cagaatggcg tgcagctctc ccaggtggat ctggtggggg
     4801 atgctgccgt tgtcgaaggt ccgctgcttc cgcagcaggt cctctctgtt cagcttcacg
     4861 agcagttcct cggtgccgtc catcttttcc aggatgggct tgatgaactt gtagaactct
     4921 tcctggctgg ctccgccgtc aatgtagccg gcgtagccgt tcttgctctg gtcgaagaaa
     4981 atctctttgt acttctcagg cagctgctgc cgcacgagag ctttcagcag ggtcaggtcc
     5041 tggtggtgct cgtcgtatct cttgatcata gaggcgctca ggggggcctt ggtgatctcg
     5101 gtgttcactc tcaggatgtc gctcagcagg atggcgtcgg acaggttctt ggcggccaga
     5161 aacaggtcgg cgtactggtc gccgatctgg gccagcaggt tgtccaggtc gtcgtcgtag
     5221 gtgtccttgc tcagctgcag tttggcatcc tcggccaggt cgaagttgct cttgaagttg
     5281 ggggtcaggc ccaggctcag ggcaatcagg tttccgaaca ggccattctt cttctcgccg
     5341 ggcagctggg cgatcagatt ttccagccgt ctgctcttgc tcagtctggc agacaggatg
     5401 gccttggcgt ccacgccgct ggcgttgatg gggttttcct cgaacagctg gttgtaggtc
     5461 tgcaccagct ggatgaacag cttgtccacg tcgctgttgt cggggttcag gtcgccctcg
     5521 atcaggaagt ggccccggaa cttgatcatg tgggccaggg ccagatagat cagccgcagg
     5581 tcggccttgt cggtgctgtc caccagtttc tttctcaggt ggtagatggt ggggtacttc
     5641 tcgtggtagg ccacctcgtc cacgatgttg ccgaagatgg ggtgccgctc gtgcttctta
     5701 tcctcttcca ccaggaagga ctcttccagt ctgtggaaga agctgtcgtc caccttggcc
     5761 atctcgttgc tgaagatctc ttgcagatag cagatccggt tcttccgtct ggtgtatctt
     5821 cttctggcgg ttctcttcag ccgggtggcc tcggctgttt cgccgctgtc gaacagcagg
     5881 gctccgatca ggttcttctt gatgctgtgc cggtcggtgt tgcccagcac cttgaatttc
     5941 ttgctgggca ccttgtactc gtcggtgatc acggcccagc ccacagagtt ggtgccgatg
     6001 gccaggccga tgctgtactt cttgtcgctg ccgccgctgc tgccgccact gctctctggt
     6061 gttgctgact cgcttgttcc tggtgtctcg ctgccagagg atcctccgct agatcctcca
     6121 gacttcaggc ctgtggccca caggatgtgg ggtggcagcc tctgatagtg acaagactgc
     6181 agggcgattg taaagaaggt cagctggggc tgctttctcc gcaggatatt cagacaaggg
     6241 ggcaggccca ggatgatgca atacagctcc agcacgtaca ggcgcaccca caggtgtggg
     6301 tacctaggcc agtgggcctc gttgctagga gaataattca cgaagttccg ccagcagtag
     6361 ccggactcct gctctgtcat gatctggatg gtcacgccgg agctgatcag atcgcgcagg
     6421 ccctgcctat tccttgggtc tgcgtggtga tacagcctgg cgatgtagat aaacagtgtc
     6481 acgtgtggat atctggacag gaactcggtg atggccctgc tacactctcc gcaaggggac
     6541 cagctcagaa accatgtgat gctacatctg gtattggggc agaagtaccg ctctgtggta
     6601 aacttctcga tgaagttcac ctccacgtgc ttatttgtgt tctgagaggt gtgcctccag
     6661 atggagtgcc tgcctcccca gttgatctcg tacagcaggc atgtctcctt ggccagctcc
     6721 cgggggtcaa agaacacttc aaactcgtga ggttcaatcc ggcggcgcag ggttggatcg
     6781 acggcgacag gcccagtctc tgaggagctc cccccgctcc cgccgctccc gccgctctcg
     6841 ctctcggcgg gcaacacggg catccagtca atgggggtct cgccccgttg ttccagccac
     6901 tgattggcca gcacgaaatg gttgcagccg aagaagcccc ggtgggcgct aagggggctg
     6961 gggtgggggg ctttcagcac atgatgccgc tgcttgtcga tgatggcgcc tttcttctgg
     7021 gcatggctgc cccacagcag aaacaccacg ccctcccggt gctggttgat caggctgatc
     7081 accttatcgg tgaaggtctc ccagcccagg ctggcgtggc tatgcgcctg gccggcccgt
     7141 accgtcagca cggtgttgag cagcagcacg ccctgccgcg cccagctctc caggtagccg
     7201 tgattgggcc gggtgaagcc ggggatggtg ttttccagct ccttatacat gttcagcagg
     7261 ctggggggga tggcgatgcc gggccgcacg ctgaaggcca gaccatgggc ctggccgggg
     7321 ccgtggtagg ggtcctggcc caggatcacc accttcacgt cgcccagctc tgtgaagcgg
     7381 aaggcgttaa acacgtcttt ctgtgggggg tagatggtca cgccgctctg ccgctcgctg
     7441 gccacggtct gcagggtgtt aaggaaatag ggctgctgct tctcttcggc cagcacgtcg
     7501 tgccaggtca gttcgttggc gactttccgc ttcttctttg gtgactcgaa ctcgcttccg
     7561 tcggctgtcc gtttcatggt ggcggctctc cctatagtga gtcgtattag cggccgcgga
     7621 tctctagcgg atctgacggt tcactaaacc agctctgctt atatagacct cccaccgtac
     7681 acgcctaccg cccatttgcg tcaatggggc ggagttgtta cgacattttg gaaagtcccg
     7741 ttgattttgg tgccaaaaca aactcccatt gacgtcaatg gggtggagac ttggaaatcc
     7801 ccgtgagtca aaccgctatc cacgcccatt gatgtactgc caaaaccgca tcaccatggt
     7861 aatagcgatg actaatacgt agatgtactg ccaagtagga aagtcccata aggtcatgta
     7921 ctgggcataa tgccaggcgg gccatttacc gtcattgacg tcaatagggg gcgtacttgg
     7981 catatgatac acttgatgta ctgccaagtg ggcagtttac cgtaaatact ccacccattg
     8041 acgtcaatgg aaagtcccta ttggcgttac tatgggaaca tacgtcatta ttgacgtcaa
     8101 tgggcggggg tcgttgggcg gtcagccagg cgggccattt accgtaagtt atgtaacgcg
     8161 gaactccata tatgggctat gaactaatga ccccgtaatt gattactatt aataactagt
     8221 caataatcaa tgtcaacgcg tatatctggc ccgtacatcg cgaagcagcg caaaacgcct
     8281 aaccctaagc agattcttca tgcaattgtc ggtcaagcct tgccttgttg tagcttaaat
     8341 tttgctcgcg cactactcag cgacctccaa cacacaagca gggagcagat actggcttaa
     8401 ctatgcggca tcagagcaga ttgtactgag agtcgaccct aggggatcgg gagatcgatc
     8461 tcccgatccg tcgacgtcag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
     8521 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     8581 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     8641 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     8701 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     8761 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     8821 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     8881 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     8941 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     9001 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     9061 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     9121 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     9181 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     9241 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     9301 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     9361 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     9421 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     9481 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     9541 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     9601 ctgagtgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     9661 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     9721 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     9781 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     9841 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     9901 tcttaccgg
//
