LOCUS       sequence-348325-        2706 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 2706)
  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 2706)
  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: pTAG4
FEATURES             Location/Qualifiers
     source          1..2706
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_RNA        3..78
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     protein_bind    208..229
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(244..249,250..267,268..274)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    282..298
                     /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     287..309
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     306..322
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     306..322
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             318..707
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGATSRLR
                     FRARNSNETTKCRGTNPVDISLFGS"
     primer_bind     341..361
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        343..361
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(408..427)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(409..427)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(437..454)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(437..453)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(446..468)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             946..1737
                     /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      2078..2666
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2567..2586
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tggtctcaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
       61 gtggcaccga gtcggtgcaa caaagcacca gtggtctagt ggtagaatag taccctgcca
      121 cggtacagac ccgggttcga ttcccggctg gtgcatgaga cgagatctag acgaccggtg
      181 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
      241 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
      301 tcacacagga aacagctatg accatgatta cgccaagcgc gcaattaacc ctcactaaag
      361 ggaacaaaag ctggaggacc tcgagggggg gcccggtacc caattcgccc tatagtgagt
      421 cgtattacgc gcgctcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
      481 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
      541 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgggacgcgc
      601 cctgtagcgg cgccactagt cgtctccgtt ttagagctag aaatagcaac gagaccacga
      661 agtgccgagg cacgaaccca gtggacataa gcctgttcgg ttcgtaagct gtaatgcaag
      721 tagcgtatgc gctcacgcaa ctggtccaga accttgaccg aacgcagcgg tggtaacggc
      781 gcagtggcgg ttttcatggc ttgttatgac tgtttttttg gggtacagtc tatgcctcgg
      841 gcatccaagc agcaagcgcg ttacgccgtg ggtcgatgtt tgatgttatg gagcagcaac
      901 gatgttacgc agcagggcag tcgccctaaa acaaagttaa acatcatggg ggaagcggtg
      961 atcgccgaag tatcgactca actatcagag gtagttggcg tcatcgagcg ccatctcgaa
     1021 ccgacgttgc tggccgtaca tttgtacggc tccgcagtgg atggcggcct gaagccacac
     1081 agcgatattg atttgctggt tacggtgacc gtaaggcttg atgaaacaac gcggcgagct
     1141 ttgatcaacg accttttgga aacttcggct tcccctggag agagcgagat tctccgcgct
     1201 gtagaagtca ccattgttgt gcacgacgac atcattccgt ggcgttatcc agctaagcgc
     1261 gaactgcaat ttggagaatg gcagcgcaat gacattcttg caggtatctt cgagccagcc
     1321 acgatcgaca ttgatctggc tatcttgctg acaaaagcaa gagaacatag cgttgccttg
     1381 gtaggtccag cggcggagga actctttgat ccggttcctg aacaggatct atttgaggcg
     1441 ctaaatgaaa ccttaacgct atggaactcg ccgcccgact gggctggcga tgagcgaaat
     1501 gtagtgctta cgttgtcccg catttggtac agcgcagtaa ccggcaaaat cgcgccgaag
     1561 gatgtcgctg ccgactgggc aatggagcgc ctgccggccc agtatcagcc cgtcatactt
     1621 gaagctagac aggcttatct tggacaagaa gaagatcgct tggcctcgcg cgcagatcag
     1681 ttggaagaat ttgtccatta cgtgaaaggc gagatcacca aggtagtcgg caaataatgt
     1741 ctagctagaa attcgttcaa gccgacgccg cttcgcggcg cggcttaact caagcgttag
     1801 atgcactaag cacataattg ctcacagcca aactatcagg tcaagtctgc ttttattatt
     1861 tttaagcgtg cataataagc cctacacaaa ttgggagata tatcatgctg tcagaccaag
     1921 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     1981 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     2041 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2101 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     2161 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     2221 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     2281 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     2341 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     2401 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     2461 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     2521 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     2581 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     2641 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgc
     2701 actctg
//
