LOCUS       sequence-348324-        2810 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 2810)
  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 2810)
  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: pTAG6E
FEATURES             Location/Qualifiers
     source          1..2810
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    176..197
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(212..217,218..235,236..242)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    250..266
                     /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     255..277
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     274..290
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     274..290
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             286..627
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEWDAPCSGATSRLR
                     FRARNSKLK"
     primer_bind     309..329
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        311..329
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(376..395)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(377..395)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(405..422)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(405..421)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(414..436)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     misc_RNA        596..671
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             1049..1840
                     /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      2181..2769
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2670..2689
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ggtctcagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgcaaca aagcaccagt
       61 ggtctagtgg tagaatagta ccctgccacg gtacagaccc gggttcgatt cccggctggt
      121 gcatgagacg agatctagac gaccggtgga aagcgggcag tgagcgcaac gcaattaatg
      181 tgagttagct cactcattag gcaccccagg ctttacactt tatgcttccg gctcgtatgt
      241 tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac catgattacg
      301 ccaagcgcgc aattaaccct cactaaaggg aacaaaagct ggaggacctc gagggggggc
      361 ccggtaccca attcgcccta tagtgagtcg tattacgcgc gctcactggc cgtcgtttta
      421 caacgtcgtg actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc
      481 cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg
      541 cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg ccactagtcg tctccgtttt
      601 agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa aaagtggcac
      661 cgagtcggtg caacaaagca ccagtggtct agtggtagaa tagtaccctg ccacggtaca
      721 gacccgggtt cgattcccgg ctggtgcagc ttcgagacca cgaagtgccg aggcacgaac
      781 ccagtggaca taagcctgtt cggttcgtaa gctgtaatgc aagtagcgta tgcgctcacg
      841 caactggtcc agaaccttga ccgaacgcag cggtggtaac ggcgcagtgg cggttttcat
      901 ggcttgttat gactgttttt ttggggtaca gtctatgcct cgggcatcca agcagcaagc
      961 gcgttacgcc gtgggtcgat gtttgatgtt atggagcagc aacgatgtta cgcagcaggg
     1021 cagtcgccct aaaacaaagt taaacatcat gggggaagcg gtgatcgccg aagtatcgac
     1081 tcaactatca gaggtagttg gcgtcatcga gcgccatctc gaaccgacgt tgctggccgt
     1141 acatttgtac ggctccgcag tggatggcgg cctgaagcca cacagcgata ttgatttgct
     1201 ggttacggtg accgtaaggc ttgatgaaac aacgcggcga gctttgatca acgacctttt
     1261 ggaaacttcg gcttcccctg gagagagcga gattctccgc gctgtagaag tcaccattgt
     1321 tgtgcacgac gacatcattc cgtggcgtta tccagctaag cgcgaactgc aatttggaga
     1381 atggcagcgc aatgacattc ttgcaggtat cttcgagcca gccacgatcg acattgatct
     1441 ggctatcttg ctgacaaaag caagagaaca tagcgttgcc ttggtaggtc cagcggcgga
     1501 ggaactcttt gatccggttc ctgaacagga tctatttgag gcgctaaatg aaaccttaac
     1561 gctatggaac tcgccgcccg actgggctgg cgatgagcga aatgtagtgc ttacgttgtc
     1621 ccgcatttgg tacagcgcag taaccggcaa aatcgcgccg aaggatgtcg ctgccgactg
     1681 ggcaatggag cgcctgccgg cccagtatca gcccgtcata cttgaagcta gacaggctta
     1741 tcttggacaa gaagaagatc gcttggcctc gcgcgcagat cagttggaag aatttgtcca
     1801 ttacgtgaaa ggcgagatca ccaaggtagt cggcaaataa tgtctagcta gaaattcgtt
     1861 caagccgacg ccgcttcgcg gcgcggctta actcaagcgt tagatgcact aagcacataa
     1921 ttgctcacag ccaaactatc aggtcaagtc tgcttttatt atttttaagc gtgcataata
     1981 agccctacac aaattgggag atatatcatg ctgtcagacc aagtttactc atatatactt
     2041 tagattgatt taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat
     2101 aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2161 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2221 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2281 tttccgaagg taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag
     2341 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2401 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2461 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2521 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     2581 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     2641 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     2701 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     2761 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tgcactctgt
//
