LOCUS       sequence-353591-        2841 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  First sgRNA in a tRNA separated array of sgRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2841)
  AUTHORS   Cody JP, Maher MF, Nasti RA, Starker CG, Chamness JC, Voytas DF
  TITLE     Direct delivery and fast-treated Agrobacterium co-culture 
            (Fast-TrACC) plant transformation methods for Nicotiana benthamiana.
  JOURNAL   Nat Protoc. 2022 Oct 17. pii: 10.1038/s41596-022-00749-9. doi: 
            10.1038/s41596-022-00749-9.
  PUBMED    36253612
REFERENCE   2  (bases 1 to 2841)
  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 Protoc.
            2022 Oct 17. pii: 10.1038/s41596-022-00749-9. doi: 
            10.1038/s41596-022-00749-9."
COMMENT     Sequence Label: pTAG4E
FEATURES             Location/Qualifiers
     source          1..2841
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    207..228
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(243..248,249..266,267..273)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    281..297
                     /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)."
     primer_bind     286..308
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     305..321
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     305..321
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             317..658
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGATSRLR
                     FRARNSKLK"
     primer_bind     340..360
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        342..360
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(407..426)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(408..426)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(436..453)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(436..452)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(445..467)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_RNA        627..702
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1080..1871
                     /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"
     rep_origin      2212..2800
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2701..2720
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggtctcagag ctagaaatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag
       61 tggcaccgag tcggtgcaac aaagcaccag tggtctagtg gtagaatagt accctgccac
      121 ggtacagacc cgggttcgat tcccggctgg tgcatgagac gagatctaga cgaccggtgg
      181 aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag
      241 gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt
      301 cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg
      361 gaacaaaagc tggaggacct cgaggggggg cccggtaccc aattcgccct atagtgagtc
      421 gtattacgcg cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt
      481 tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga
      541 ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc
      601 ctgtagcggc gccactagtc gtctccgttt tagagctaga aatagcaagt taaaataagg
      661 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gcaacaaagc accagtggtc
      721 tagtggtaga atagtaccct gccacggtac agacccgggt tcgattcccg gctggtgcag
      781 cttcgagacc acgaagtgcc gaggcacgaa cccagtggac ataagcctgt tcggttcgta
      841 agctgtaatg caagtagcgt atgcgctcac gcaactggtc cagaaccttg accgaacgca
      901 gcggtggtaa cggcgcagtg gcggttttca tggcttgtta tgactgtttt tttggggtac
      961 agtctatgcc tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt
     1021 tatggagcag caacgatgtt acgcagcagg gcagtcgccc taaaacaaag ttaaacatca
     1081 tgggggaagc ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt ggcgtcatcg
     1141 agcgccatct cgaaccgacg ttgctggccg tacatttgta cggctccgca gtggatggcg
     1201 gcctgaagcc acacagcgat attgatttgc tggttacggt gaccgtaagg cttgatgaaa
     1261 caacgcggcg agctttgatc aacgaccttt tggaaacttc ggcttcccct ggagagagcg
     1321 agattctccg cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt ccgtggcgtt
     1381 atccagctaa gcgcgaactg caatttggag aatggcagcg caatgacatt cttgcaggta
     1441 tcttcgagcc agccacgatc gacattgatc tggctatctt gctgacaaaa gcaagagaac
     1501 atagcgttgc cttggtaggt ccagcggcgg aggaactctt tgatccggtt cctgaacagg
     1561 atctatttga ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc gactgggctg
     1621 gcgatgagcg aaatgtagtg cttacgttgt cccgcatttg gtacagcgca gtaaccggca
     1681 aaatcgcgcc gaaggatgtc gctgccgact gggcaatgga gcgcctgccg gcccagtatc
     1741 agcccgtcat acttgaagct agacaggctt atcttggaca agaagaagat cgcttggcct
     1801 cgcgcgcaga tcagttggaa gaatttgtcc attacgtgaa aggcgagatc accaaggtag
     1861 tcggcaaata atgtctagct agaaattcgt tcaagccgac gccgcttcgc ggcgcggctt
     1921 aactcaagcg ttagatgcac taagcacata attgctcaca gccaaactat caggtcaagt
     1981 ctgcttttat tatttttaag cgtgcataat aagccctaca caaattggga gatatatcat
     2041 gctgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     2101 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     2161 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2221 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2281 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2341 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2401 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2461 cgataagtcg tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg
     2521 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2581 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     2641 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     2701 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     2761 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     2821 tttacggttc ctgcactctg t
//
