LOCUS       sequence-322131-        4216 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 4216)
  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 4216)
  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: pAT9658-sgRNA-mCherry
FEATURES             Location/Qualifiers
     source          1..4216
                     /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"
     regulatory      1466..1475
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1472..2182
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(1617..1635)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     complement(2212..2229)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2218..2425
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(2490..3078)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2570..2589)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3249..4040,4041..4109))
                     /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     3872..3891
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4110..4214)
                     /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 catggtgagc aagggcgagg aggataacat
     1501 ggccatcatc aaggagttca tgcgcttcaa ggtgcacatg gagggctccg tgaacggcca
     1561 cgagttcgag atcgagggcg agggcgaggg ccgcccctac gagggcaccc agaccgccaa
     1621 gctgaaggtg accaagggtg gccccctgcc cttcgcctgg gacatcctgt cccctcagtt
     1681 catgtacggc tccaaggcct acgtgaagca ccccgccgac atccccgact acttgaagct
     1741 gtccttcccc gagggcttca agtgggagcg cgtgatgaac ttcgaggacg gcggcgtggt
     1801 gaccgtgacc caggactcct ccctgcagga cggcgagttc atctacaagg tgaagctgcg
     1861 cggcaccaac ttcccctccg acggccccgt aatgcagaaa aagaccatgg gctgggaggc
     1921 ctcctccgag cggatgtacc ccgaggacgg cgccctgaag ggcgagatca agcagaggct
     1981 gaagctgaag gacggcggcc actacgacgc tgaggtcaag accacctaca aggccaagaa
     2041 gcccgtgcag ctgcccggcg cctacaacgt caacatcaag ttggacatca cctcccacaa
     2101 cgaggactac accatcgtgg aacagtacga acgcgccgag ggccgccact ccaccggcgg
     2161 catggacgag ctgtacaagt agatctacat gtcctagagc tcgctgatca gcctcgactg
     2221 tgccttctag ttgccagcca tctgttgttt gcccctcccc cgtgccttcc ttgaccctgg
     2281 aaggtgccac tcccactgtc ctttcctaat aaaatgagga aattgcatcg cattgtctga
     2341 gtaggtgtca ttctattctg gggggtgggg tggggcagga cagcaagggg gaggattggg
     2401 aagagaatag caggcatgct ggggagcggc cgctgagcaa aaggccagca aaaggccagg
     2461 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     2521 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     2581 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     2641 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     2701 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     2761 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     2821 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     2881 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     2941 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     3001 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     3061 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     3121 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     3181 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     3241 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     3301 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     3361 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     3421 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     3481 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     3541 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     3601 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     3661 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     3721 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     3781 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     3841 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     3901 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     3961 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     4021 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     4081 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     4141 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     4201 ataggggttc cgcgca
//
