LOCUS       Exported                3004 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Prime editing in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pU6-pegRNA-GG-acceptor
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3004)
  AUTHORS   Anzalone AV, Randolph PB, Davis JR, Sousa AA, Koblan LW, Levy JM, 
            Chen PJ, Wilson C, Newby GA, Raguram A, Liu DR
  TITLE     Search-and-replace genome editing without double-strand breaks or 
            donor DNA.
  JOURNAL   Nature. 2019 Oct 21. pii: 10.1038/s41586-019-1711-4. doi: 
            10.1038/s41586-019-1711-4.
  PUBMED    31634902
REFERENCE   2  (bases 1 to 3004)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..3004
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        82..322
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA"
     primer_bind     82..102
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     253..272
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     terminator      340..371
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(388..1065)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             1072..1083
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    1101..1119
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1126..1144
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     rep_origin      complement(1255..1843)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1335..1354)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(2014..2874)
                     /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     2637..2656
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2875..2979)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacgtcgcta gctgtacaaa aaagcaggct ttaaaggaac caattcagtc gactggatcc
       61 ggtaccaagg tcgggcagga agagggccta tttcccatga ttccttcata tttgcatata
      121 cgatacaagg ctgttagaga gataattaga attaatttga ctgtaaacac aaagatatta
      181 gtacaaaata cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta
      241 tgttttaaaa tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct
      301 ttatatatct tgtggaaagg acgaaacacc cgagaccgta gtcaaaagcc tccggtcgga
      361 ggcttttgac ttggctgagg agtgccatta agcaccggtg gagtgacgac cttcagcacg
      421 ttcgtactgt tcaacgatgg tgtagtcttc gttgtgggag gtgatgtcca gtttgatgtc
      481 ggttttgtaa gcacccggca gctgaaccgg ttttttagcc atgtaggtgg ttttaacttc
      541 agcgtcgtag tgaccaccgt ctttcagttt cagacgcatt ttgatttcac ctttcagagc
      601 accgtcttcc gggtacatac gttcggtgga agcttcccaa cccatggttt ttttctgcat
      661 aaccggaccg tcggacggga agttggtacc acgcagttta actttgtaga tgaactcacc
      721 gtcttgcagg gaggagtcct gggtaacggt aacaacacca ccgtcttcga agttcataac
      781 acgttcccat ttgaaacctt ccgggaagga cagtttcagg tagtccggga tgtcagccgg
      841 gtgtttaacg taagctttgg aaccgtactg gaactgcggg gacaggatgt cccaagcgaa
      901 cggcagcgga ccacctttgg taactttcag tttagcggtc tgggtacctt cgtacggacg
      961 accttcacct tcaccttcga tttcgaactc gtgaccgtta acggaacctt ccatacgaac
     1021 tttgaaacgc atgaactctt tgataacgtc ttcgctactc gccatagatc ctttctcctc
     1081 tttcagatcc gtgctcagta tctctatcac tgatagggat gtcaatctct atcactgata
     1141 gggaggtctc gtttttttaa gcttgggccg ctcgaggtac ctctctacat atgacatgtg
     1201 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     1261 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     1321 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     1381 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     1441 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     1501 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     1561 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     1621 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     1681 cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     1741 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     1801 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     1861 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     1921 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     1981 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     2041 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     2101 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagatcc
     2161 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     2221 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     2281 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     2341 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     2401 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     2461 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     2521 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     2581 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     2641 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     2701 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     2761 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     2821 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     2881 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     2941 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     3001 acct
//
