LOCUS       SaBE4-Gam                8601 bp ds-DNA     circular SYN 06-AUG-2018
DEFINITION  Expresses SaBE4-Gam in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    SaBE4-Gam
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8601)
  AUTHORS   Komor AC, Zhao KT, Packer MS, Gaudelli NM, Waterbury AL, Koblan LW, 
            Kim YB, Badran AH, Liu DR
  TITLE     Improved base excision repair inhibition and bacteriophage Mu Gam 
            protein yields C:G-to-T:A base editors with higher efficiency and 
            product purity.
  JOURNAL   Sci Adv. 2017 Aug 30;3(8):eaao4774. doi: 10.1126/sciadv.aao4774. 
            eCollection 2017 Aug.
  PUBMED    28875174
REFERENCE   2  (bases 1 to 8601)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 6, 2018 from SnapGene Server 1.1.58
            //www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8601
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        132..335
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     286..306
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     377..396
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        377..395
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      403..412
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             979..1662
                     /codon_start=1
                     /product="cytidine deaminase (C to U editing enzyme) from 
                     rat"
                     /label=APOBEC-1
                     /note="can use ssDNA as a substrate (Komor et al., 2016)"
                     /translation="SSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWGG
                     RHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFLS
                     RYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSNE
                     AHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHIL
                     WATGLK"
     CDS             4924..4944
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4951..5016
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             5047..5295
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5326..5574
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5587..5607
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5616..5633
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5656..5673)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5662..5886
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5957..5973)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5957..5973)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5970..5992)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5981..5997
                     /label=lac operator
                     /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)."
     promoter        complement(6005..6035)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6050..6071
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6188..6205)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6359..6947)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6439..6458)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7118..7978)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     7741..7760
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7979..8083)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8162..8181)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        8353..131
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
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 ggctaaacca
      421 gcaaaacgta tcaagagtgc cgcagcggct tatgtgccac aaaaccgcga tgcggtgatt
      481 accgatatta aacgcatcgg ggatttacag cgcgaagcat cacgtctgga aacggaaatg
      541 aatgatgcca tcgcggaaat tacggagaaa tttgcggccc ggattgcacc gattaaaacc
      601 gatattgaaa ccctttcaaa aggcgttcag ggatggtgtg aagcgaaccg cgacgaactg
      661 acgaacggcg gcaaagtgaa gacggcgaat cttgtcaccg gtgatgtatc gtggcgggtc
      721 cgtccaccat cagtaagtat tcgtggtatg gatgcagtga tggaaacgct ggagcgtctt
      781 ggcctgcaac gctttattcg cacgaagcag gaaatcaaca aggaagcgat tttactggaa
      841 ccgaaagcgg tcgcaggcgt tgccggaatt acagttaaat caggcattga ggatttttct
      901 attattccat ttgaacagga agccggtatt agcggcagcg agactcccgg gacctcagag
      961 tccgctacac ccgaaagtag ctcagagact ggcccagtgg ctgtggaccc cacattgaga
     1021 cggcggatcg agccccatga gtttgaggta ttcttcgatc cgagagagct ccgcaaggag
     1081 acctgcctgc tttacgaaat taattggggg ggccggcact ccatttggcg acatacatca
     1141 cagaacacta acaagcacgt cgaagtcaac ttcatcgaga agttcacgac agaaagatat
     1201 ttctgtccga acacaaggtg cagcattacc tggtttctca gctggagccc atgcggcgaa
     1261 tgtagtaggg ccatcactga attcctgtca aggtatcccc acgtcactct gtttatttac
     1321 atcgcaaggc tgtaccacca cgctgacccc cgcaatcgac aaggcctgcg ggatttgatc
     1381 tcttcaggtg tgactatcca aattatgact gagcaggagt caggatactg ctggagaaac
     1441 tttgtgaatt atagcccgag taatgaagcc cactggccta ggtatcccca tctgtgggta
     1501 cgactgtacg ttcttgaact gtactgcatc atactgggcc tgcctccttg tctcaacatt
     1561 ctgagaagga agcagccaca gctgacattc tttaccatcg ctcttcagtc ttgtcattac
     1621 cagcgactgc ccccacacat tctctgggcc accgggttga aatctggtgg ttcttctggt
     1681 ggttctagcg gcagcgagac tcccgggacc tcagagtccg ccacacccga aagttctggt
     1741 ggttcttctg gtggttctgg gaaacggaac tacatcctgg ggcttgccat tgggataacc
     1801 agcgttggct acggaattat tgattatgag acacgcgatg tgattgacgc cggggttagg
     1861 ctgttcaaag aggccaacgt tgaaaacaac gagggaagac ggagtaagcg cggagcaaga
     1921 agactcaagc gcagacggag acatcggatt cagagggtga aaaagctgct cttcgattac
     1981 aatctcctga ccgatcatag tgagctgagc ggaatcaacc cctacgaggc gcgagtgaaa
     2041 gggctttccc agaagctgtc cgaagaggag ttctccgccg cgttgctgca cctggccaaa
     2101 cggagggggg ttcacaatgt aaacgaagtg gaggaggaca cgggcaatga acttagtacg
     2161 aaagaacaga tcagtaggaa ctctaaggct ctcgaagaga aatacgtcgc tgagttgcag
     2221 cttgagagac tgaaaaaaga cggcgaagta cgcggatcta ttaataggtt caagacttca
     2281 gattacgtaa aggaagccaa gcagctcctg aaagtacaga aagcgtacca tcagctcgat
     2341 cagagcttca tcgataccta catagatttg ctggagacac ggaggacata ctacgagggc
     2401 ccaggggaag gatctccttt tgggtggaag gacatcaagg aatggtacga gatgcttatg
     2461 ggacattgta catattttcc ggaggagctc aggagcgtca agtacgccta caatgccgac
     2521 ctgtacaatg ccctcaatga cctcaataac ctcgtgatta ccagggacga gaacgagaag
     2581 ctggagtact atgaaaagtt ccagattatc gagaatgtgt ttaagcagaa gaagaagccg
     2641 acacttaagc agattgcaaa ggaaatcctc gtgaatgagg aagatatcaa gggatacaga
     2701 gtgacaagta caggcaagcc cgagttcaca aatctgaagg tgtaccacga tattaaggac
     2761 ataaccgcac gaaaggagat aatcgaaaac gctgagctcc tcgatcagat cgcaaaaatt
     2821 cttaccatct accagtctag tgaggacatt caggaggaac tgactaatct gaacagtgag
     2881 ctcacccaag aggaaattga gcagatttca aacctgaaag gctacaccgg gacgcacaat
     2941 ctgagcctca aagcaatcaa cctcattctg gatgaacttt ggcacacaaa tgacaaccaa
     3001 attgccatat tcaaccgcct gaaactggtg ccaaaaaaag tggatctgtc acagcaaaag
     3061 gaaatcccta caaccttggt tgacgatttt attctgtccc ccgttgtcaa gcggagcttc
     3121 atccagtcaa tcaaggtgat caatgccatc attaaaaaat acggattgcc aaacgatata
     3181 attatcgagc ttgcacgaga gaagaactca aaggacgccc agaagatgat taacgaaatg
     3241 cagaagcgca accgccagac aaacgaacgc atagaggaaa ttataagaac aaccggcaaa
     3301 gagaatgcca agtatctgat cgagaaaatc aagctgcacg acatgcaaga aggcaagtgc
     3361 ctgtactctc tggaagctat cccactcgaa gatctgctga ataatccatt caattacgag
     3421 gtggaccaca tcatccctag atccgtaagc tttgacaatt ccttcaataa caaagttctg
     3481 gttaaacagg aggaaaattc taaaaaaggg aaccggaccc cgttccagta cctgagctcc
     3541 agtgacagca agattagcta cgagactttt aagaaacata ttctgaatct ggccaaaggc
     3601 aaaggcagga tcagcaagac caagaaggag tacctcctcg aagaacgcga cattaacaga
     3661 tttagtgtgc agaaagattt catcaaccga aaccttgtcg atactcggta cgccacgaga
     3721 ggcctgatga atctcctcag gagctacttc cgcgtcaata atctggacgt taaagtcaag
     3781 agcataaatg ggggattcac cagctttctg aggagaaagt ggaagtttaa gaaggaacga
     3841 aacaaaggat acaagcacca tgctgaggat gctttgatca tcgctaacgc ggactttatc
     3901 tttaaggaat ggaaaaagct ggataaggca aagaaagtga tggaaaacca gatgttcgag
     3961 gagaagcagg cagagtcaat gcctgagatc gagacagagc aggaatacaa ggaaattttc
     4021 atcacccctc atcagattaa acacataaag gacttcaaag actataaata ctctcatagg
     4081 gtggacaaaa aacccaatcg cgagctcatt aatgacaccc tgtactcaac acggaaggat
     4141 gataaaggta ataccttgat tgtgaataat cttaatggat tgtatgacaa agataacgac
     4201 aagctcaaga agctgatcaa caagtctcca gagaagctcc ttatgtatca ccacgaccca
     4261 cagacttatc agaaattgaa actgatcatg gagcaatacg gggatgagaa gaacccactc
     4321 tacaaatatt atgaggaaac aggtaattac ctgaccaagt actccaagaa ggataacgga
     4381 ccagtgatca aaaagataaa gtactatggc aacaaactta atgcgcattt ggacataact
     4441 gacgattacc ccaattctcg aaacaaggtt gtgaagctct ccctgaagcc ttatagattt
     4501 gacgtgtacc tggataatgg ggtttataaa ttcgtcaccg tgaaaaatct ggacgtgatc
     4561 aaaaaggaga actattatga agtaaactca aagtgctatg aggaggcgaa gaagctgaag
     4621 aagatctcca atcaggccga gttcatcgct tccttctata ataacgatct catcaagatc
     4681 aatggagagc tttatcgcgt cattggtgtg aacaatgact tgctgaacag gatcgaagtc
     4741 aatatgatag acattaccta ccgggagtat ctcgaaaaca tgaatgataa acggccgcct
     4801 cgcatcatca agacaatcgc atctaaaact cagtcaataa aaaagtactc taccgatatc
     4861 ctggggaatc tctatgaagt gaagtcaaag aagcacccac aaatcattaa aaaaggtgga
     4921 tcccccaaga agaagaggaa agtctcgagc gactacaaag accatgacgg tgattataaa
     4981 gatcatgaca tcgattacaa ggatgacgat gacaagtctg gtggttctgg aggatctggt
     5041 ggttctacta atctgtcaga tattattgaa aaggagaccg gtaagcaact ggttatccag
     5101 gaatccatcc tcatgctccc agaggaggtg gaagaagtca ttgggaacaa gccggaaagc
     5161 gatatactcg tgcacaccgc ctacgacgag agcaccgacg agaatgtcat gcttctgact
     5221 agcgacgccc ctgaatacaa gccttgggct ctggtcatac aggatagcaa cggtgagaac
     5281 aagattaaga tgctctctgg tggttctgga ggatctggtg gttctactaa tctgtcagat
     5341 attattgaaa aggagaccgg taagcaactg gttatccagg aatccatcct catgctccca
     5401 gaggaggtgg aagaagtcat tgggaacaag ccggaaagcg atatactcgt gcacaccgcc
     5461 tacgacgaga gcaccgacga gaatgtcatg cttctgacta gcgacgcccc tgaatacaag
     5521 ccttgggctc tggtcataca ggatagcaac ggtgagaaca agattaagat gctctctggt
     5581 ggttctccca agaagaagag gaaagtctaa ccggtcatca tcaccatcac cattgagttt
     5641 aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt gtttgcccct
     5701 cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc taataaaatg
     5761 aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt ggggtggggc
     5821 aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat gcggtgggct
     5881 ctatggcttc tgaggcggaa agaaccagct ggggctcgat accgtcgacc tctagctaga
     5941 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     6001 cacacaacat acgagccgga agcataaagt gtaaagccta gggtgcctaa tgagtgagct
     6061 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     6121 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     6181 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     6241 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     6301 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     6361 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     6421 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     6481 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     6541 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     6601 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     6661 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     6721 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     6781 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     6841 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     6901 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     6961 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     7021 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     7081 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     7141 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     7201 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     7261 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     7321 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     7381 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     7441 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     7501 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     7561 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     7621 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     7681 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     7741 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     7801 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     7861 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     7921 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     7981 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     8041 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     8101 tgccacctga cgtcgacgga tcgggagatc gatctcccga tcccctaggg tcgactctca
     8161 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     8221 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     8281 attgcatgaa gaatctgctt agggttaggc gttttgcgct gcttcgcgat gtacgggcca
     8341 gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt acggggtcat
     8401 tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat ggcccgcctg
     8461 gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt cccatagtaa
     8521 cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa actgcccact
     8581 tggcagtaca tcaagtgtat c
//