LOCUS       pCMV_BE1_YTH_HA         4726 bp ds-DNA     circular     10-SEP-2019
DEFINITION  Expresses BE1 with C-terminal NLS in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pCMV-BE1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8271)
  AUTHORS   Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR
  TITLE     Programmable editing of a target base in genomic DNA without
            double-stranded DNA cleavage.
  JOURNAL   Nature. 2016 Apr 20. doi: 10.1038/nature17946.
  PUBMED    27096365
REFERENCE   2  (bases 1 to 8271)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 10, 2018 from SnapGene Server 1.1.58
            http://www.snapgene.com
COMMENT     
COMMENT     
COMMENT     ApEinfo:methylated:1
FEATURES             Location/Qualifiers
     source          1..1111
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /label=source:synthetic DNA construct
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    1141..1215
                     /locus_tag="Beginning of m6A binding region (385-579)"
                     /label=Beginning of m6A binding region (385-579)
                     /ApEinfo_fwdcolor="#ff80c0"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     promoter        132..335
                     /note="human cytomegalovirus (CMV) immediate early
                     promoter"
                     /label=CMV promoter
                     /ApEinfo_fwdcolor="#346ee0"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    1141..1143
                     /locus_tag="In frame with protein"
                     /label=In frame with protein
                     /ApEinfo_fwdcolor="#ff80ff"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     primer_bind     286..306
                     /note="Human CMV immediate early promoter, forward primer"
                     /label=CMV-F
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     source          1112..1135
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /locus_tag="source:synthetic DNA construct"
                     /label=source:synthetic DNA construct(1)
                     /ApEinfo_label="source:synthetic DNA construct"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     primer_bind     377..396
                     /note="T7 promoter, forward primer"
                     /label=T7
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     source          1762..1765
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /locus_tag="source:synthetic DNA construct(1)"
                     /label=source:synthetic DNA construct(2)
                     /ApEinfo_label="source:synthetic DNA construct"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     promoter        377..395
                     /note="promoter for bacteriophage T7 RNA polymerase"
                     /label=T7 promoter
                     /ApEinfo_fwdcolor="#346ee0"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             409..1095
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from
                     rat"
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINW
                     G
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEF
                     L
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPS
                     N
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPH
                     I LWATGLK"
                     /label=APOBEC-1
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     primer_bind     complement(1781..1798)
                     /note="Bovine growth hormone terminator, reverse primer.
                     Also called BGH reverse"
                     /label=BGH-rev
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     polyA_signal    1787..2011
                     /note="bovine growth hormone polyadenylation signal"
                     /label=bGH poly(A) signal
                     /ApEinfo_fwdcolor="#ff3eee"
                     /ApEinfo_revcolor="#ff3eee"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     primer_bind     complement(2082..2098)
                     /note="common sequencing primer, one of multiple similar
                     variants"
                     /label=M13 rev
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     primer_bind     complement(2082..2098)
                     /note="In lacZ gene. Also called M13-rev"
                     /label=M13 Reverse
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     primer_bind     complement(2095..2117)
                     /note="In lacZ gene"
                     /label=M13/pUC Reverse
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     protein_bind    2106..2122
                     /bound_moiety="lac repressor encoded by lacI"
                     /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)."
                     /label=lac operator
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     promoter        complement(2130..2160)
                     /note="promoter for the E. coli lac operon"
                     /label=lac promoter
                     /ApEinfo_fwdcolor="#346ee0"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     protein_bind    2175..2196
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence
                     of cAMP."
                     /label=CAP binding site
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     primer_bind     complement(2313..2330)
                     /note="L4440 vector, forward primer"
                     /label=L4440
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     rep_origin      complement(2484..3072)
                     /direction=LEFT
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
                     replication"
                     /label=ori
                     /ApEinfo_fwdcolor="#999999"
                     /ApEinfo_revcolor="#999999"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     primer_bind     complement(2564..2583)
                     /note="pBR322 origin, forward primer"
                     /label=pBR322ori-F
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             complement(3243..4103)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /note="confers resistance to ampicillin, carbenicillin,
                     and related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     I
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEY
                     S
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDR
                     W
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRS
                     A
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGA
                     S LIKHW"
                     /label=AmpR
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     primer_bind     3866..3885
                     /note="Ampicillin resistance gene, reverse primer"
                     /label=Amp-R
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     promoter        complement(4104..4208)
                     /gene="bla"
                     /label=AmpR promoter
                     /ApEinfo_fwdcolor="#346ee0"
                     /ApEinfo_revcolor="#346ee0"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     primer_bind     complement(4287..4306)
                     /note="pRS vectors, use to sequence yeast selectable
                     marker"
                     /label=pRS-marker
                     /ApEinfo_fwdcolor="#14c0bd"
                     /ApEinfo_revcolor="#4ec02b"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     enhancer        join(4478..4726,1..131)
                     /note="human cytomegalovirus immediate early enhancer"
                     /label=CMV enhancer
                     /ApEinfo_fwdcolor="#5ac3fa"
                     /ApEinfo_revcolor="#5ac3fa"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     source          1766..4726
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
                     /label=source:synthetic DNA construct(3)
                     /ApEinfo_label="source:synthetic DNA construct"
                     /ApEinfo_fwdcolor="pink"
                     /ApEinfo_revcolor="pink"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     misc_feature    1216..1725
                     /locus_tag="YTH Domain"
                     /label=YTH Domain
                     /ApEinfo_fwdcolor="cyan"
                     /ApEinfo_revcolor="green"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
     CDS             1732..1758
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /locus_tag="HA"
                     /label=HA
                     /ApEinfo_fwdcolor="#e9d024"
                     /ApEinfo_revcolor="#e9d024"
                     /ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
                     width 5 offset 0"
ORIGIN
        1 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
       61 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      121 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      181 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      241 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      301 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      361 agagatccgc ggccgctaat acgactcact atagggagag ccgccaccat gagctcagag
      421 actggcccag tggctgtgga ccccacattg agacggcgga tcgagcccca tgagtttgag
      481 gtattcttcg atccgagaga gctccgcaag gagacctgcc tgctttacga aattaattgg
      541 gggggccggc actccatttg gcgacataca tcacagaaca ctaacaagca cgtcgaagtc
      601 aacttcatcg agaagttcac gacagaaaga tatttctgtc cgaacacaag gtgcagcatt
      661 acctggtttc tcagctggag cccatgcggc gaatgtagta gggccatcac tgaattcctg
      721 tcaaggtatc cccacgtcac tctgtttatt tacatcgcaa ggctgtacca ccacgctgac
      781 ccccgcaatc gacaaggcct gcgggatttg atctcttcag gtgtgactat ccaaattatg
      841 actgagcagg agtcaggata ctgctggaga aactttgtga attatagccc gagtaatgaa
      901 gcccactggc ctaggtatcc ccatctgtgg gtacgactgt acgttcttga actgtactgc
      961 atcatactgg gcctgcctcc ttgtctcaac attctgagaa ggaagcagcc acagctgaca
     1021 ttctttacca tcgctcttca gtcttgtcat taccagcgac tgcccccaca cattctctgg
     1081 gccaccgggt tgaaaagcgg cagcgagact cccgggacct cagagtccgc cacaccagaa
     1141 ccccacccag tgttggagaa gcttcggtcc attaataact ataaccccaa agattttgac
     1201 tggaatctga aacatGGCCG GGTTTTCATC ATTAAGAGCT ACTCTGAGGA CGATATTCAC
     1261 CGTTCCATTA AGTATAATAT TTGGTGCAGC ACAGAGCATG GTAACAAGAG ACTGGATGCT
     1321 GCTTATCGTT CCATGAACGG GAAAGGCCCC GTTTACTTAC TTTTCAGTGT CAACGGCAGT
     1381 GGACACTTCT GTGGCGTGGC AGAAATGAAA TCTGCTGTGG ACTACAACAC ATGTGCAGGT
     1441 GTGTGGTCCC AGGACAAATG GAAGGGTCGT TTTGATGTCA GGTGGATTTT TGTGAAGGAC
     1501 GTTCCCAATA GCCAACTGCG ACACATTCGC CTAGAGAACA ACGAGAATAA ACCAGTGACC
     1561 AACTCTAGGG ACACTCAGGA AGTGCCTCTG GAAAAGGCTA AGCAGGTGTT GAAAATTATA
     1621 GCCAGCTACA AGCACACCAC TTCCATTTTT GATGACTTCT CACACTATGA GAAACGCCAA
     1681 GAGGAAGAAG AAAGTGTTAA AAAGGAACGT CAAGGTCGTG GGAAActcga gtacccctac
     1741 gacgtgcccg actacgcctg agtttaaacc cgctgatcag cctcgactgt gccttctagt
     1801 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     1861 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     1921 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     1981 aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac cagctggggc
     2041 tcgataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct gtttcctgtg
     2101 tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat aaagtgtaaa
     2161 gcctagggtg cctaatgagt gagctaactc acattaattg cgttgcgctc actgcccgct
     2221 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     2281 ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct gcgctcggtc
     2341 gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt atccacagaa
     2401 tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt
     2461 aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga gcatcacaaa
     2521 aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata ccaggcgttt
     2581 ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac cggatacctg
     2641 tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg taggtatctc
     2701 agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc
     2761 gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag acacgactta
     2821 tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt aggcggtgct
     2881 acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt atttggtatc
     2941 tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg atccggcaaa
     3001 caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac gcgcagaaaa
     3061 aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa
     3121 aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt
     3181 ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac
     3241 agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc
     3301 atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt accatctggc
     3361 cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata
     3421 aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc
     3481 cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc
     3541 aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca
     3601 ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa
     3661 gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca
     3721 ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt
     3781 tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt
     3841 tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg
     3901 ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga
     3961 tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc
     4021 agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg
     4081 acacggaaat gttgaatact catactcttc ctttttcaat attattgaag catttatcag
     4141 ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg
     4201 gttccgcgca catttccccg aaaagtgcca cctgacgtcg acggatcggg agatcgatct
     4261 cccgatcccc tagggtcgac tctcagtaca atctgctctg atgccgcata gttaagccag
     4321 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
     4381 acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt
     4441 gcgctgcttc gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat
     4501 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
     4561 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
     4621 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
     4681 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatc 
//
