LOCUS       Exported                8034 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses SaBE4 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    SaBE4
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8034)
  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 8034)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8034
                     /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"
     CDS             409..1095
                     /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="MSSETGPVAVDPTLRRRIEPHEFEVFFDPRELRKETCLLYEINWG
                     GRHSIWRHTSQNTNKHVEVNFIEKFTTERYFCPNTRCSITWFLSWSPCGECSRAITEFL
                     SRYPHVTLFIYIARLYHHADPRNRQGLRDLISSGVTIQIMTEQESGYCWRNFVNYSPSN
                     EAHWPRYPHLWVRLYVLELYCIILGLPPCLNILRRKQPQLTFFTIALQSCHYQRLPPHI
                     LWATGLK"
     CDS             4357..4377
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             4384..4449
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags, followed by an
                     enterokinase cleavage site"
                     /label=3xFLAG
                     /translation="DYKDHDGDYKDHDIDYKDDDDK"
     CDS             4480..4728
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             4759..5007
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5020..5040
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5049..5066
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5089..5106)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5095..5319
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5390..5406)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5390..5406)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5403..5425)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5414..5430
                     /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(5438..5468)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5483..5504
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5621..5638)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5792..6380)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5872..5891)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6551..7411)
                     /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     7174..7193
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7412..7516)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7595..7614)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        7786..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 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 tgaaatctgg tggttcttct ggtggttcta gcggcagcga gactcccggg
     1141 acctcagagt ccgccacacc cgaaagttct ggtggttctt ctggtggttc tgggaaacgg
     1201 aactacatcc tggggcttgc cattgggata accagcgttg gctacggaat tattgattat
     1261 gagacacgcg atgtgattga cgccggggtt aggctgttca aagaggccaa cgttgaaaac
     1321 aacgagggaa gacggagtaa gcgcggagca agaagactca agcgcagacg gagacatcgg
     1381 attcagaggg tgaaaaagct gctcttcgat tacaatctcc tgaccgatca tagtgagctg
     1441 agcggaatca acccctacga ggcgcgagtg aaagggcttt cccagaagct gtccgaagag
     1501 gagttctccg ccgcgttgct gcacctggcc aaacggaggg gggttcacaa tgtaaacgaa
     1561 gtggaggagg acacgggcaa tgaacttagt acgaaagaac agatcagtag gaactctaag
     1621 gctctcgaag agaaatacgt cgctgagttg cagcttgaga gactgaaaaa agacggcgaa
     1681 gtacgcggat ctattaatag gttcaagact tcagattacg taaaggaagc caagcagctc
     1741 ctgaaagtac agaaagcgta ccatcagctc gatcagagct tcatcgatac ctacatagat
     1801 ttgctggaga cacggaggac atactacgag ggcccagggg aaggatctcc ttttgggtgg
     1861 aaggacatca aggaatggta cgagatgctt atgggacatt gtacatattt tccggaggag
     1921 ctcaggagcg tcaagtacgc ctacaatgcc gacctgtaca atgccctcaa tgacctcaat
     1981 aacctcgtga ttaccaggga cgagaacgag aagctggagt actatgaaaa gttccagatt
     2041 atcgagaatg tgtttaagca gaagaagaag ccgacactta agcagattgc aaaggaaatc
     2101 ctcgtgaatg aggaagatat caagggatac agagtgacaa gtacaggcaa gcccgagttc
     2161 acaaatctga aggtgtacca cgatattaag gacataaccg cacgaaagga gataatcgaa
     2221 aacgctgagc tcctcgatca gatcgcaaaa attcttacca tctaccagtc tagtgaggac
     2281 attcaggagg aactgactaa tctgaacagt gagctcaccc aagaggaaat tgagcagatt
     2341 tcaaacctga aaggctacac cgggacgcac aatctgagcc tcaaagcaat caacctcatt
     2401 ctggatgaac tttggcacac aaatgacaac caaattgcca tattcaaccg cctgaaactg
     2461 gtgccaaaaa aagtggatct gtcacagcaa aaggaaatcc ctacaacctt ggttgacgat
     2521 tttattctgt cccccgttgt caagcggagc ttcatccagt caatcaaggt gatcaatgcc
     2581 atcattaaaa aatacggatt gccaaacgat ataattatcg agcttgcacg agagaagaac
     2641 tcaaaggacg cccagaagat gattaacgaa atgcagaagc gcaaccgcca gacaaacgaa
     2701 cgcatagagg aaattataag aacaaccggc aaagagaatg ccaagtatct gatcgagaaa
     2761 atcaagctgc acgacatgca agaaggcaag tgcctgtact ctctggaagc tatcccactc
     2821 gaagatctgc tgaataatcc attcaattac gaggtggacc acatcatccc tagatccgta
     2881 agctttgaca attccttcaa taacaaagtt ctggttaaac aggaggaaaa ttctaaaaaa
     2941 gggaaccgga ccccgttcca gtacctgagc tccagtgaca gcaagattag ctacgagact
     3001 tttaagaaac atattctgaa tctggccaaa ggcaaaggca ggatcagcaa gaccaagaag
     3061 gagtacctcc tcgaagaacg cgacattaac agatttagtg tgcagaaaga tttcatcaac
     3121 cgaaaccttg tcgatactcg gtacgccacg agaggcctga tgaatctcct caggagctac
     3181 ttccgcgtca ataatctgga cgttaaagtc aagagcataa atgggggatt caccagcttt
     3241 ctgaggagaa agtggaagtt taagaaggaa cgaaacaaag gatacaagca ccatgctgag
     3301 gatgctttga tcatcgctaa cgcggacttt atctttaagg aatggaaaaa gctggataag
     3361 gcaaagaaag tgatggaaaa ccagatgttc gaggagaagc aggcagagtc aatgcctgag
     3421 atcgagacag agcaggaata caaggaaatt ttcatcaccc ctcatcagat taaacacata
     3481 aaggacttca aagactataa atactctcat agggtggaca aaaaacccaa tcgcgagctc
     3541 attaatgaca ccctgtactc aacacggaag gatgataaag gtaatacctt gattgtgaat
     3601 aatcttaatg gattgtatga caaagataac gacaagctca agaagctgat caacaagtct
     3661 ccagagaagc tccttatgta tcaccacgac ccacagactt atcagaaatt gaaactgatc
     3721 atggagcaat acggggatga gaagaaccca ctctacaaat attatgagga aacaggtaat
     3781 tacctgacca agtactccaa gaaggataac ggaccagtga tcaaaaagat aaagtactat
     3841 ggcaacaaac ttaatgcgca tttggacata actgacgatt accccaattc tcgaaacaag
     3901 gttgtgaagc tctccctgaa gccttataga tttgacgtgt acctggataa tggggtttat
     3961 aaattcgtca ccgtgaaaaa tctggacgtg atcaaaaagg agaactatta tgaagtaaac
     4021 tcaaagtgct atgaggaggc gaagaagctg aagaagatct ccaatcaggc cgagttcatc
     4081 gcttccttct ataataacga tctcatcaag atcaatggag agctttatcg cgtcattggt
     4141 gtgaacaatg acttgctgaa caggatcgaa gtcaatatga tagacattac ctaccgggag
     4201 tatctcgaaa acatgaatga taaacggccg cctcgcatca tcaagacaat cgcatctaaa
     4261 actcagtcaa taaaaaagta ctctaccgat atcctgggga atctctatga agtgaagtca
     4321 aagaagcacc cacaaatcat taaaaaaggt ggatccccca agaagaagag gaaagtctcg
     4381 agcgactaca aagaccatga cggtgattat aaagatcatg acatcgatta caaggatgac
     4441 gatgacaagt ctggtggttc tggaggatct ggtggttcta ctaatctgtc agatattatt
     4501 gaaaaggaga ccggtaagca actggttatc caggaatcca tcctcatgct cccagaggag
     4561 gtggaagaag tcattgggaa caagccggaa agcgatatac tcgtgcacac cgcctacgac
     4621 gagagcaccg acgagaatgt catgcttctg actagcgacg cccctgaata caagccttgg
     4681 gctctggtca tacaggatag caacggtgag aacaagatta agatgctctc tggtggttct
     4741 ggaggatctg gtggttctac taatctgtca gatattattg aaaaggagac cggtaagcaa
     4801 ctggttatcc aggaatccat cctcatgctc ccagaggagg tggaagaagt cattgggaac
     4861 aagccggaaa gcgatatact cgtgcacacc gcctacgacg agagcaccga cgagaatgtc
     4921 atgcttctga ctagcgacgc ccctgaatac aagccttggg ctctggtcat acaggatagc
     4981 aacggtgaga acaagattaa gatgctctct ggtggttctc ccaagaagaa gaggaaagtc
     5041 taaccggtca tcatcaccat caccattgag tttaaacccg ctgatcagcc tcgactgtgc
     5101 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     5161 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     5221 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     5281 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     5341 gctggggctc gataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     5401 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5461 agtgtaaagc ctagggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5521 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5581 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5641 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5701 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5761 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5821 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5881 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5941 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6001 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6061 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6121 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6181 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     6241 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6301 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6361 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6421 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6481 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6541 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6601 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6661 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6721 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6781 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6841 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6901 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6961 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7021 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7081 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     7141 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     7201 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7261 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7321 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7381 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7441 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7501 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac ggatcgggag
     7561 atcgatctcc cgatccccta gggtcgactc tcagtacaat ctgctctgat gccgcatagt
     7621 taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc gcgagcaaaa
     7681 tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg cttagggtta
     7741 ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat tgattattga
     7801 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     7861 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     7921 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     7981 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatc
//
