LOCUS       sequence-391249-        3594 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Golden Gate entry vector; 2nd gRNA under OsU3 promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3594)
  AUTHORS   Lee K, Kang M, Ji Q, Grosic S, Wang K
  TITLE     New T-DNA binary vectors with NptII selection and RUBY reporter for 
            efficient maize transformation and targeted mutagenesis.
  JOURNAL   Plant Physiol. 2023 Apr 18:kiad231. doi: 10.1093/plphys/kiad231.
  PUBMED    37070560
REFERENCE   2  (bases 1 to 3594)
  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: "Plant 
            Physiol. 2023 Apr 18:kiad231. doi: 10.1093/plphys/kiad231."
COMMENT     Sequence Label: pCBLsgRNA2
FEATURES             Location/Qualifiers
     source          1..3594
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(1..19)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(24..40)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(24..40)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        129..506
                     /label=OsU3 promoter
                     /note="Oryza sativa (rice) snRNA U3 promoter"
     rep_origin      807..1395
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1296..1315
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     terminator      complement(1725..1752)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(1844..1930)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     promoter        join(2154..2159,2160..2176,2177..2182)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     CDS             2230..3420
                     /codon_start=1
                     /gene="tet"
                     /product="tetracycline efflux protein"
                     /label=TcR
                     /note="confers resistance to tetracycline"
                     /translation="MKSNNALIVILGTVTLDAVGIGLVMPVLPGLLRDIVHSDSIASHY
                     GVLLALYALMQFLCAPVLGALSDRFGRRPVLLASLLGATIDYAIMATTPVLWILYAGRI
                     VAGITGATGAVAGAYIADITDGEDRARHFGLMSACFGVGMVAGPVAGGLLGAISLHAPF
                     LAAAVLNGLNLLLGCFLMQESHKGERRPMPLRAFNPVSSFRWARGMTIVAALMTVFFIM
                     QLVGQVPAALWVIFGEDRFRWSATMIGLSLAVFGILHALAQAFVTGPATKRFGEKQAII
                     AGMAADALGYVLLAFATRGWMAFPIMILLASGGIGMPALQAMLSRQVDDDHQGQLQGSL
                     AALTSLTSIIGPLIVTAIYAASASTWNGLAWIVGAALYLVCLPALRRGAWSRATST"
     primer_bind     complement(2582..2601)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     complement(join(3594,1..19))
                     /label=T7
                     /note="T7 promoter, forward primer"
ORIGIN
        1 cctatagtga gtcgtattac atggtcatag ctgtttcctg gcagctctgg cccgtgtctc
       61 aaaatctctg atgttacatt gcacaagata aaaatatatc atcatgcctc ctctagaggt
      121 ctcccatgaa gggatcttta aacatacgaa cagatcactt aaagttcttc tgaagcaact
      181 taaagttatc aggcatgcat ggatcttgga ggaatcagat gtgcagtcag ggaccatagc
      241 acaggacagg cgtcttctac tggtgctacc agcaaatgct ggaagccggg aacactgggt
      301 acgttggaaa ccacgtgatg tggagtaaga taaactgtag gagaaaagca tttcgtagtg
      361 ggccatgaag cctttcagga catgtattgc agtatgggcc ggcccattac gcaattggac
      421 gacaacaaag actagtatta gtaccacctc ggctatccac atagatcaaa gctggtttaa
      481 aagagttgtg cagatgatcc gtggcagaga cgagatctag tctgagtcga ccgtctctgt
      541 ttcagagcta tgctggaaac agcatagcaa gttgaaataa ggctagtccg ttatcaactt
      601 gaaaaagtgg caccgagtcg gtgctttttt tgtcccttcg aagggcaatt ctgcagatat
      661 ccatcacact ggcggccgct cgaggtcgag ggtatcgata agcttggacc gagaccctcg
      721 agccacccat gaccaaaatc ccttaacgtg agttacgcgt cgttccactg agcgtcagac
      781 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
      841 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
      901 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
      961 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     1021 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     1081 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     1141 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     1201 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     1261 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     1321 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     1381 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     1441 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     1501 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     1561 agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt
     1621 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     1681 attaatacgc gtaccgctag ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca
     1741 ggatggcctt ctgcttagtt tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc
     1801 ctccgggccg ttgcttcaca acgttcaaat ccgctcccgg cggatttgtc ctactcagga
     1861 gagcgttcac cgacaaacaa cagataaaac gaaaggccca gtcttccgac tgagcctttc
     1921 gttttatttg atgcctggca gttccctact ctcgcgttct gcattaatga atcggccaac
     1981 gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc
     2041 tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt
     2101 tatccacaga atcaggggat aacgcaggaa agaacatgaa ttaattctca tgtttgacag
     2161 cttatcatcg attagcttta atgcggtagt ttatcacagt taaattgcta acgcagtcag
     2221 gcaccgtgta tgaaatctaa caatgcgctc atcgtcatcc tcggcaccgt caccctggat
     2281 gctgtaggca taggcttggt tatgccggta ctgccgggcc tcttgcggga tatcgtccat
     2341 tccgacagca tcgccagtca ctatggcgtg ctgctagcgc tatatgcgtt gatgcaattt
     2401 ctatgcgcac ccgttctcgg agcactgtcc gaccgctttg gccgccgccc agtcctgctc
     2461 gcttcgctac ttggagccac tatcgactac gcgatcatgg cgaccacacc cgtcctgtgg
     2521 attctctacg ccggacgcat cgtggccggc atcaccggcg ccacaggtgc ggttgctggc
     2581 gcctatatcg ccgacatcac cgatggggaa gatcgggctc gccacttcgg gctcatgagc
     2641 gcttgtttcg gcgtgggtat ggtggcaggc cccgtggccg ggggactgtt gggcgccatc
     2701 tccttacatg caccattcct tgcggcggcg gtgctcaacg gcctcaacct actactgggc
     2761 tgcttcctaa tgcaggagtc gcataaggga gagcgccgac ccatgccctt gagagccttc
     2821 aacccagtca gctccttccg gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact
     2881 gtcttcttta tcatgcaact cgtaggacag gtgccggcag cgctctgggt cattttcggc
     2941 gaggaccgct ttcgctggag cgcgacgatg atcggcctgt cgcttgcggt attcggaatc
     3001 ttgcacgccc tcgctcaagc cttcgtcact ggtcccgcca ccaaacgttt cggcgagaag
     3061 caggccatta tcgccggcat ggcggccgac gcgctgggct acgtcttgct ggcgttcgcg
     3121 acgcgaggct ggatggcctt ccccattatg attcttctcg cttccggcgg catcgggatg
     3181 cccgcgttgc aggccatgct gtccaggcag gtagatgacg accatcaggg acagcttcaa
     3241 ggatcgctcg cggctcttac cagcctaact tcgatcattg gaccgctgat cgtcacggcg
     3301 atttatgccg cctcggcgag cacatggaac gggttggcat ggattgtagg cgccgcccta
     3361 taccttgtct gcctccccgc gttgcgtcgc ggtgcatgga gccgggccac ctcgacctga
     3421 atggaagccg gcggcacctc gctaacggat tcaccactcc aagaattgga gccaatcaat
     3481 tcttgcggag aactgtgaat gcgcaaacca acccttgatc ggggaagaac agtatgtcga
     3541 gctatttttt gacttactgg ggatcaagcc tgattgggag aaaataaaat atcc
//
