LOCUS       sequence-348329-        2831 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 2831)
  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 2831)
  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: pTAG2E
FEATURES             Location/Qualifiers
     source          1..2831
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    197..218
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(233..238,239..256,257..263)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    271..287
                     /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     276..298
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     295..311
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     295..311
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             307..648
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGATSRLR
                     FRARNSKLK"
     primer_bind     330..350
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        332..350
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(397..416)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(398..416)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(426..443)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(426..442)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(435..457)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_RNA        617..692
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1070..1861
                     /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      2202..2790
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2691..2710
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggtctcatag caagttaaaa taaggctagt ccgttatcaa cttgaaaaag tggcaccgag
       61 tcggtgcaac aaagcaccag tggtctagtg gtagaatagt accctgccac ggtacagacc
      121 cgggttcgat tcccggctgg tgcatgagac gagatctaga cgaccggtgg aaagcgggca
      181 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
      241 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
      301 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
      361 tggaggacct cgaggggggg cccggtaccc aattcgccct atagtgagtc gtattacgcg
      421 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
      481 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
      541 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
      601 gccactagtc gtctccgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
      661 atcaacttga aaaagtggca ccgagtcggt gcaacaaagc accagtggtc tagtggtaga
      721 atagtaccct gccacggtac agacccgggt tcgattcccg gctggtgcag cttcgagacc
      781 acgaagtgcc gaggcacgaa cccagtggac ataagcctgt tcggttcgta agctgtaatg
      841 caagtagcgt atgcgctcac gcaactggtc cagaaccttg accgaacgca gcggtggtaa
      901 cggcgcagtg gcggttttca tggcttgtta tgactgtttt tttggggtac agtctatgcc
      961 tcgggcatcc aagcagcaag cgcgttacgc cgtgggtcga tgtttgatgt tatggagcag
     1021 caacgatgtt acgcagcagg gcagtcgccc taaaacaaag ttaaacatca tgggggaagc
     1081 ggtgatcgcc gaagtatcga ctcaactatc agaggtagtt ggcgtcatcg agcgccatct
     1141 cgaaccgacg ttgctggccg tacatttgta cggctccgca gtggatggcg gcctgaagcc
     1201 acacagcgat attgatttgc tggttacggt gaccgtaagg cttgatgaaa caacgcggcg
     1261 agctttgatc aacgaccttt tggaaacttc ggcttcccct ggagagagcg agattctccg
     1321 cgctgtagaa gtcaccattg ttgtgcacga cgacatcatt ccgtggcgtt atccagctaa
     1381 gcgcgaactg caatttggag aatggcagcg caatgacatt cttgcaggta tcttcgagcc
     1441 agccacgatc gacattgatc tggctatctt gctgacaaaa gcaagagaac atagcgttgc
     1501 cttggtaggt ccagcggcgg aggaactctt tgatccggtt cctgaacagg atctatttga
     1561 ggcgctaaat gaaaccttaa cgctatggaa ctcgccgccc gactgggctg gcgatgagcg
     1621 aaatgtagtg cttacgttgt cccgcatttg gtacagcgca gtaaccggca aaatcgcgcc
     1681 gaaggatgtc gctgccgact gggcaatgga gcgcctgccg gcccagtatc agcccgtcat
     1741 acttgaagct agacaggctt atcttggaca agaagaagat cgcttggcct cgcgcgcaga
     1801 tcagttggaa gaatttgtcc attacgtgaa aggcgagatc accaaggtag tcggcaaata
     1861 atgtctagct agaaattcgt tcaagccgac gccgcttcgc ggcgcggctt aactcaagcg
     1921 ttagatgcac taagcacata attgctcaca gccaaactat caggtcaagt ctgcttttat
     1981 tatttttaag cgtgcataat aagccctaca caaattggga gatatatcat gctgtcagac
     2041 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     2101 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     2161 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     2221 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     2281 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     2341 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     2401 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     2461 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     2521 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     2581 ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc ggacaggtat
     2641 ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg gggaaacgcc
     2701 tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg atttttgtga
     2761 tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt tttacggttc
     2821 ctgcactctg t
//
