LOCUS       sequence-322132-        4208 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA cloning plasmid .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4208)
  AUTHORS   Talas A, Simon DA, Kulcsar PI, Varga E, Krausz SL, Welker E
  TITLE     BEAR reveals that increased fidelity variants can successfully 
            reduce the mismatch tolerance of adenine but not cytosine base 
            editors.
  JOURNAL   Nat Commun. 2021 Nov 3;12(1):6353. doi: 10.1038/s41467-021-26461-y.
  PUBMED    34732717
REFERENCE   2  (bases 1 to 4208)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat 
            Commun."; date: "2021-11-3"; volume: "12(1):6353. doi"; pages: " 
            10.1038/s41467-021-26461-y"
COMMENT     Sequence Label: pAT9679-sgRNA-BFP
FEATURES             Location/Qualifiers
     source          1..4208
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        53..301
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     53..73
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     224..243
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     terminator      complement(312..343)
                     /label=tonB terminator
                     /note="bidirectional E. coli tonB-P14 transcription 
                     terminator"
     CDS             complement(360..1037)
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     RBS             1044..1055
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     protein_bind    1073..1091
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1098..1116
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     misc_RNA        1125..1200
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        1235..1446
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     CDS             1472..2173
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     primer_bind     complement(2204..2221)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2210..2417
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2482..3070)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2562..2581)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3241..4032,4033..4101))
                     /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     3864..3883
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4102..4206)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 catttccccg aaaagtgcca cctgacgtcc agcagagatc cactttggcg ccgagggcct
       61 atttcccatg attccttcat atttgcatat acgatacaag gctgttagag agataattgg
      121 aattaatttg actgtaaaca caaagatatt agtacaaaat acgtgacgta gaaagtaata
      181 atttcttggg tagtttgcag ttttaaaatt atgttttaaa atggactatc atatgcttac
      241 cgtaacttga aagtatttcg atttcttggc tttatatatc ttgtggaaag gacgaaacac
      301 cgggtcttcg tagtcaaaag cctccggtcg gaggcttttg acttggctga ggagtgccat
      361 taagcaccgg tggagtgacg accttcagca cgttcgtact gttcaacgat ggtgtaatct
      421 tcgttgtggg aggtgatgtc cagtttgatg tcggttttgt aagcacccgg cagctgaacc
      481 ggttttttag ccatgtaggt ggttttaact tcagcgtcgt agtgaccacc gtctttcagt
      541 ttcagacgca ttttgatttc acctttcaga gcaccatctt ccgggtacat acgttcggtg
      601 gaagcttccc aacccatggt ttttttctgc ataaccggac cgtcggacgg gaagttggta
      661 ccacgcagtt taactttgta gatgaactca ccgtcttgca gggaggagtc ctgggtaacg
      721 gtaacaacac caccatcttc gaagttcata acacgttccc atttgaaacc ttccgggaag
      781 gacagtttca ggtagtccgg gatgtcagcc gggtgtttaa cgtaagcttt ggaaccgtac
      841 tggaactgcg gggacaggat gtcccaagcg aacggcagcg gaccaccttt ggtaactttc
      901 agtttagcgg tctgggtacc ttcgtacgga cgaccttcac cttcaccttc gatttcgaac
      961 tcgtgaccgt taacggaacc ttccatacga actttgaaac gcatgaactc tttgataaca
     1021 tcttcgctac tcgccataga tcctttctcc tctttcagat ccgtgctcag tatctctatc
     1081 actgataggg atgtcaatct ctatcactga tagggagaag acctgtttta gagctagaaa
     1141 tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc
     1201 ttttttggct cgagtggctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc
     1261 cccgagaagt tggggggagg ggtcggcaat tgaaccggtg cctagagaag gtggcgcggg
     1321 gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa
     1381 ccgtatataa gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga
     1441 acacaggtgt cgtgacgcgg gatccgccac catgagcgag ctgattaagg agaacatgca
     1501 catgaagctg tacatggagg gcaccgtgga caaccatcac ttcaagtgca catccgaggg
     1561 cgaaggcaag ccctacgagg gcacccagac catgagaatc aaggtggtcg agggcggccc
     1621 tctccccttc gccttcgaca tcctggctac tagcttcctc tacggcagca agaccttcat
     1681 caaccacacc cagggcatcc ccgacttctt caagcagtcc ttccctgagg gcttcacatg
     1741 ggagagagtc accacatacg aagatggggg cgtgctgacc gctacccagg acaccagcct
     1801 ccaggacggc tgcctcatct acaacgtcaa gatcagaggg gtgaacttca catccaacgg
     1861 ccctgtgatg cagaagaaaa cactcggctg ggaggccttc accgagacgc tgtaccccgc
     1921 tgacggcggc ctggaaggca gaaacgacat ggccctgaag ctcgtgggcg ggagccatct
     1981 gatcgcaaac atcaagacca catatagatc caagaaaccc gctaagaacc tcaagatgcc
     2041 tggcgtctac tatgtggact acagactgga aagaatcaag gaggccaaca acgagaccta
     2101 cgtcgagcag cacgaggtgg cagtggccag atactgcgac ctccctagca aactggggca
     2161 caagcttaat taagatctac atgtcctaga gctcgctgat cagcctcgac tgtgccttct
     2221 agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc
     2281 actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt
     2341 cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagagaat
     2401 agcaggcatg ctggggagcg gccgctgagc aaaaggccag caaaaggcca ggaaccgtaa
     2461 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     2521 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     2581 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     2641 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     2701 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     2761 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     2821 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     2881 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     2941 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     3001 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     3061 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     3121 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     3181 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     3241 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     3301 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     3361 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     3421 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     3481 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     3541 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     3601 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     3661 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     3721 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     3781 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     3841 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     3901 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     3961 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     4021 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     4081 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     4141 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     4201 tccgcgca
//
