LOCUS       sequence-232091-        4629 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of a target sgRNA in potato.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4629)
  AUTHORS   Zong Y, Song Q, Li C, Jin S, Zhang D, Wang Y, Qiu JL, Gao C
  TITLE     Efficient C-to-T base editing in plants using a fusion of nCas9 and 
            human APOBEC3A.
  JOURNAL   Nat Biotechnol. 2018 Oct 1. pii: nbt.4261. doi: 10.1038/nbt.4261.
  PUBMED    30272679
REFERENCE   2  (bases 1 to 4629)
  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 
            Biotechnol. 2018 Oct 1. pii: nbt.4261. doi: 10.1038/nbt.4261."
COMMENT     Sequence Label: pAtU6-sgRNA
FEATURES             Location/Qualifiers
     source          1..4629
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1180..1971
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MGEAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     misc_RNA        2123..2198
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2408..2424)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2408..2424)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2421..2443)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2432..2448
                     /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(join(2456..2462,2463..2480,2481..2486))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2501..2522
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2639..2656)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2810..3398)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2890..2909)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3569..4360,4361..4429))
                     /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     4192..4211
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4430..4534)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4602..4620
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa
      421 tgcatctaga tatcggatcc cgggcccgtc gactgcagag gcctgcatgc aagcttcgac
      481 ttgccttccg cacaatacat catttcttct tagctttttt tcttcttctt cgttcataca
      541 gttttttttt gtttatcagc ttacattttc ttgaaccgta gctttcgttt tcttcttttt
      601 aactttccat tcggagtttt tgtatcttgt ttcatagttt gtcccaggat tagaatgatt
      661 aggcatcgaa ccttcaagaa tttgattgaa taaaacatct tcattcttaa gatatgaaga
      721 taatcttcaa aaggcccctg ggaatctgaa agaagagaag caggcccatt tatatgggaa
      781 agaacaatag tatttcttat ataggcccat ttaagttgaa aacaatcttc aaaagtccca
      841 catcgcttag ataagaaaac gaagctgagt ttatatacag ctagagtcga agtagtgatt
      901 gggagaccaa cccagtggac ataagcctgt tcggttcgta agctgtaatg caagtagcgt
      961 atgcgctcac gcaactggtc cagaaccttg accgaacgca gcggtggtaa cggcgcagtg
     1021 gcggttttca tggcttgtta tgactgtttt tttggggtac agtctatgcc tcgggcatcc
     1081 aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag caacgatgtt
     1141 acgcagcagg gcagtcgccc taaaacaaag ttaaacatca tgggggaagc ggtgatcgcc
     1201 gaagtatcga ctcaactatc agaggtagtt ggcgtcatcg agcgccatct cgaaccgacg
     1261 ttgctggccg tacatttgta cggctccgca gtggatggcg gcctgaagcc acacagtgat
     1321 attgatttgc tggttacggt gaccgtaagg cttgatgaaa caacgcggcg agctttgatc
     1381 aacgaccttt tggaaacttc ggcttcccct ggagagagcg agattctccg cgctgtagaa
     1441 gtcaccattg ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa gcgcgaactg
     1501 caatttggag aatggcagcg caatgacatt cttgcaggta tcttcgagcc agccacgatc
     1561 gacattgatc tggctatctt gctgacaaaa gcaagagaac atagcgttgc cttggtaggt
     1621 ccagcggcgg aggaactctt tgatccggtt cctgaacagg atctatttga ggcgctaaat
     1681 gaaaccttaa cgctatggaa ctcgccgccc gactgggctg gcgatgagcg aaatgtagtg
     1741 cttacgttgt cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc gaaggatgtc
     1801 gctgccgact gggcaatgga gcgcctgccg gcccagtatc agcccgtcat acttgaagct
     1861 agacaggctt atcttggaca agaagaagat cgcttggcct cgcgcgcaga tcagttggaa
     1921 gaatttgtcc actacgtgaa aggcgagatc accaaggtag tcggcaaata atgtctagct
     1981 agaaattcgt tcaagccgac gccgcttcgc ggcgcggctt aactcaagcg ttagatgcac
     2041 taagcacata attgctcaca gccaaactat caggtcaagt ctgcttttat tatttttaag
     2101 cgtgcataat aagccggtct cggttttaga gctagaaata gcaagttaaa ataaggctag
     2161 tccgttatca acttgaaaaa gtggcaccga gtcggtgctt ttttttgcaa aattttccag
     2221 atcgatttct tcttcctctg ttcttcggcg ttcaatttct ggggttttct cttcgttttc
     2281 tgtaactgaa acctaaaatt tgacctaaaa aaaatctcaa ataatatgat tcagtggttt
     2341 tgtacttttc agttagttga gttttgcagt tccgatgaga taaaccaata agcttggcgt
     2401 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     2461 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     2521 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     2581 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     2641 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     2701 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     2761 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     2821 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     2881 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     2941 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     3001 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     3061 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     3121 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     3181 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     3241 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     3301 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     3361 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     3421 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     3481 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     3541 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     3601 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     3661 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gaaccacgct
     3721 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     3781 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     3841 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     3901 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     3961 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     4021 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     4081 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     4141 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     4201 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     4261 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     4321 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     4381 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     4441 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     4501 gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccacctg
     4561 acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt atcacgaggc
     4621 cctttcgtc
//
