LOCUS       pCMV-dCpf1-BE                8166 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses dLbCpf1-BE in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pCMV-dCpf1-BE
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8166)
  AUTHORS   Li X, Wang Y, Liu Y, Yang B, Wang X, Wei J, Lu Z, Zhang Y, Wu J, 
            Huang X, Yang L, Chen J
  TITLE     Base editing with a Cpf1-cytidine deaminase fusion.
  JOURNAL   Nat Biotechnol. 2018 Mar 19. pii: nbt.4102. doi: 10.1038/nbt.4102.
  PUBMED    29553573
REFERENCE   2  (bases 1 to 8166)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            //gt12365.cn
FEATURES             Location/Qualifiers
     source          1..8166
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     enhancer        39..418
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        419..622
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     573..593
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     664..683
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        664..682
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             699..719
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             720..1406
                     /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             5145..5165
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5178..5426
                     /codon_start=1
                     /product="uracil-DNA glycosylase inhibitor from a Bacillus 
                     subtilis bacteriophage (Mol et al., 1995)"
                     /label=UGI
                     /translation="TNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTA
                     YDESTDENVMLLTSDAPEYKPWALVIQDSNGENKIKML"
     CDS             5439..5459
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             5468..5485
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(5508..5525)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5514..5738
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5809..5825)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5809..5825)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5822..5844)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5833..5849
                     /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(5857..5887)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5902..5923
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6040..6057)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6211..6799)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6291..6310)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6970..7830)
                     /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     7593..7612
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7831..7935)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(8014..8033)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgcgctgctt cgcgatgtac gggccagata tacgcgttga cattgattat tgactagtta
       61 ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt tccgcgttac
      121 ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc cattgacgtc
      181 aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac gtcaatgggt
      241 ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata tgccaagtac
      301 gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc agtacatgac
      361 cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta ttaccatggt
      421 gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac ggggatttcc
      481 aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc aacgggactt
      541 tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg
      601 ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctaga gatccgcggc
      661 cgctaatacg actcactata gggagagccg ccaccatgcc caagaagaag aggaaagtca
      721 tgagctcaga gactggccca gtggctgtgg accccacatt gagacggcgg atcgagcccc
      781 atgagtttga ggtattcttc gatccgagag agctccgcaa ggagacctgc ctgctttacg
      841 aaattaattg ggggggccgg cactccattt ggcgacatac atcacagaac actaacaagc
      901 acgtcgaagt caacttcatc gagaagttca cgacagaaag atatttctgt ccgaacacaa
      961 ggtgcagcat tacctggttt ctcagctgga gcccatgcgg cgaatgtagt agggccatca
     1021 ctgaattcct gtcaaggtat ccccacgtca ctctgtttat ttacatcgca aggctgtacc
     1081 accacgctga cccccgcaat cgacaaggcc tgcgggattt gatctcttca ggtgtgacta
     1141 tccaaattat gactgagcag gagtcaggat actgctggag aaactttgtg aattatagcc
     1201 cgagtaatga agcccactgg cctaggtatc cccatctgtg ggtacgactg tacgttcttg
     1261 aactgtactg catcatactg ggcctgcctc cttgtctcaa cattctgaga aggaagcagc
     1321 cacagctgac attctttacc atcgctcttc agtcttgtca ttaccagcga ctgcccccac
     1381 acattctctg ggccaccggg ttgaaaagcg gcagcgagac tcccgggacc tcagagtccg
     1441 ccacacccga aagtatgtca aagctggaga aattcaccaa ctgttatagc ctgtctaaga
     1501 ccctgcgctt caaggcaatc ccagtgggca agacacaaga gaacattgac aacaaacggc
     1561 tcctggtgga ggatgagaag agggctgaag attacaaggg cgttaagaag ctgctggata
     1621 ggtactatct gtcattcatc aacgatgtcc tccacagtat caagctgaag aatctgaaca
     1681 attacatttc tctgttccgg aagaagacac ggaccgagaa ggagaacaaa gagctggaga
     1741 atctggagat caacctgagg aaagaaatag ctaaggcttt caaagggaac gagggttaca
     1801 agtccctgtt caagaaagac attatcgaga ctattctgcc tgagttcctg gacgataaag
     1861 atgagatcgc cctcgtcaat tccttcaatg ggtttaccac agcctttacc ggcttcttcg
     1921 acaatagaga gaatatgttc tctgaagagg ccaaatccac tagcatcgcc tttcgctgca
     1981 taaacgagaa cctgactagg tacatcagca atatggacat ctttgagaaa gtcgatgcca
     2041 tattcgacaa acatgaggtg caggagatta aggagaagat cctgaactca gattacgatg
     2101 tcgaagattt cttcgaggga gagttcttca acttcgtgct cacacaagag ggcattgatg
     2161 tgtacaatgc aatcattgga gggttcgtga cagagagtgg cgagaagata aagggcctga
     2221 acgagtatat caacctctac aaccagaaaa ccaagcagaa actgcctaag ttcaagccac
     2281 tgtacaaaca agtgctctca gatagggaaa gcctgagctt ctacggtgaa gggtatacat
     2341 cagatgaaga agtgctcgaa gtgttccgca acaccctcaa taagaacagt gaaatcttct
     2401 cttcaatcaa gaagctggag aaactgttca agaatttcga tgagtactcc tctgccggaa
     2461 tctttgtgaa gaatggccct gcaatatcca ctattagcaa agacatcttt ggcgagtgga
     2521 acgttatcag ggataagtgg aatgccgagt acgatgatat tcatctcaag aagaaagccg
     2581 tggttacaga gaaatacgag gatgatagac gcaagagctt taagaagatt ggtagcttct
     2641 ctctcgaaca gctgcaggag tacgccgacg ctgacctgtc agtcgtggag aaactcaagg
     2701 agatcataat ccagaaggtg gatgaaatct acaaagtgta tggaagctct gagaaactct
     2761 tcgatgcaga ctttgttctg gagaagagtc tgaagaagaa cgacgcagtg gttgctatca
     2821 tgaaggacct gctggattct gttaagtctt tcgagaatta cattaaggca ttctttggtg
     2881 aagggaagga gacaaatagg gacgagagct tctatggcga ctttgttctg gcctacgaca
     2941 tcctcctcaa ggttgaccac atctatgacg ctatacggaa ttacgttacc cagaagccct
     3001 atagcaaaga caagttcaag ctgtatttcc agaatccaca gtttatgggt gggtgggata
     3061 aagacaaaga aacagattac agggccacta tcctgcggta cggcagcaaa tactatctgg
     3121 ctatcatgga taagaagtac gccaaatgcc tccagaagat cgacaaggac gacgtgaacg
     3181 gtaactacga gaagatcaat tacaagctcc tgccaggacc taacaagatg ctgcccaagg
     3241 tgttcttctc caagaaatgg atggcctact ataacccaag cgaggacatt cagaagatat
     3301 acaagaatgg gacattcaag aagggcgata tgttcaacct caacgactgc cacaagctga
     3361 ttgatttctt caaggatagc atttctcgct atcccaagtg gtctaatgca tacgatttca
     3421 acttcagcga gactgagaag tacaaagaca tcgctggctt ctaccgggag gtggaagagc
     3481 aaggctataa ggtgtcattc gaatccgctt ctaagaagga agtggataag ctcgtggaag
     3541 agggtaagct gtacatgttc cagatataca acaaagactt cagcgataag agccacggca
     3601 ctccaaacct ccatactatg tatttcaagc tgctgtttga cgagaacaac cacggacaga
     3661 ttaggctgtc aggaggcgca gaactcttca tgcgcagagc ttcactgaag aaggaggaac
     3721 tcgttgtcca cccagccaat agccctatag ccaataagaa tccagacaat cctaagaaaa
     3781 ccactactct gtcttacgat gtgtataagg ataagagatt ctctgaagat cagtacgaac
     3841 tgcacatacc cattgccatt aacaagtgcc ctaagaacat cttcaagatt aacacagagg
     3901 ttagagtgct cctgaaacac gacgataacc cttatgttat aggcattgct cgcggagaga
     3961 gaaacctgct gtacatcgtc gtggtggacg gcaaaggcaa catcgtggaa cagtacagtc
     4021 tcaatgaaat cattaacaat ttcaacggaa tccgcattaa gaccgactac cattctctcc
     4081 tcgacaagaa ggagaaagaa aggttcgaag caagacagaa ttggacaagt atagagaata
     4141 tcaaagaact gaaggctggg tacatctctc aggttgtgca caagatatgt gagctggtgg
     4201 agaagtacga cgctgttatc gccctcgcgg acctgaatag cggcttcaag aactccaggg
     4261 tgaaggtgga gaagcaggtg tatcagaagt tcgagaagat gctgatcgac aagctcaact
     4321 atatggtgga caagaaatcc aatccttgcg ctactggtgg agccctgaag ggctatcaaa
     4381 tcaccaataa gttcgaatct ttcaagtcta tgagcaccca gaatggcttc atcttctaca
     4441 tacccgcatg gctgacatcc aagattgatc cctctaccgg atttgttaat ctgctcaaga
     4501 ctaagtacac ctctattgct gactcaaaga agttcatatc atcatttgac cgcatcatgt
     4561 acgtgccaga agaggacctg ttcgagtttg ccctggatta caagaatttc tctcggactg
     4621 acgccgacta catcaagaag tggaagctct actcttatgg taatcggatt cgcatattcc
     4681 gcaatcccaa gaagaataac gtgttcgatt gggaggaagt ttgcctcacc agcgcttaca
     4741 aggagctgtt caataagtat gggattaact accagcaggg cgacataaga gccctgctgt
     4801 gcgaacaatc tgataaggca ttctattcct ctttcatggc actgatgtca ctgatgctgc
     4861 aaatgcgcaa ttccatcacc ggaagaacag acgtggcctt tctgatctct cctgtcaaga
     4921 actcagatgg catcttctac gattcccgca actatgaagc acaggagaat gctatcctgc
     4981 ctaagaatgc cgatgcaaat ggagcctata acatcgccag aaaggtcctc tgggccatag
     5041 gacaattcaa gaaagctgaa gatgagaagc tggacaaggt gaagatcgcc atttcaaaca
     5101 aagagtggct cgaatatgct cagacctcag tgaagcatgg atcacccaag aagaaacgga
     5161 aagtgtctgg tggttctact aatctgtcag atattattga aaaggagacc ggtaagcaac
     5221 tggttatcca ggaatccatc ctcatgctcc cagaggaggt ggaagaagtc attgggaaca
     5281 agccggaaag cgatatactc gtgcacaccg cctacgacga gagcaccgac gagaatgtca
     5341 tgcttctgac tagcgacgcc cctgaataca agccttgggc tctggtcata caggatagca
     5401 acggtgagaa caagattaag atgctctctg gtggttctcc caagaagaag aggaaagtct
     5461 aaccggtcat catcaccatc accattgagt ttaaacccgc tgatcagcct cgactgtgcc
     5521 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     5581 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     5641 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     5701 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     5761 ctggggctcg ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt
     5821 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5881 gtgtaaagcc tagggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5941 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     6001 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     6061 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     6121 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     6181 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     6241 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     6301 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     6361 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     6421 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     6481 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6541 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6601 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6661 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6721 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6781 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6841 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6901 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6961 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     7021 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     7081 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     7141 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     7201 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     7261 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     7321 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     7381 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     7441 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     7501 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7561 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7621 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7681 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7741 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7801 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7861 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7921 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga
     7981 tcgatctccc gatcccctag ggtcgactct cagtacaatc tgctctgatg ccgcatagtt
     8041 aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg cgagcaaaat
     8101 ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc ttagggttag
     8161 gcgttt
//