LOCUS       sequence-315319-        3325 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AtpU6-29 Golden Gate level 0 piece to express gRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3325)
  AUTHORS   Triozzi P, Schmidt HW, Dervinis C, Kirst M, Conde D
  TITLE     Simple, efficient and open-source CRISPR/Cas9 strategy for 
            multi-site genome editing in Populus tremula x alba.
  JOURNAL   Tree Physiol. 2021 May 7. pii: 6270869. doi: 
            10.1093/treephys/tpab066.
  PUBMED    33960379
REFERENCE   2  (bases 1 to 3325)
  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: "Tree 
            Physiol. 2021 May 7. pii: 6270869. doi: 10.1093/treephys/tpab066."
COMMENT     Sequence Label: pGEM-T Easy-AtpU6-29
FEATURES             Location/Qualifiers
     source          1..3325
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      33..42
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     regulatory      37..46
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(450..468)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(451..468)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(486..502)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(486..502)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(499..521)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    510..526
                     /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)."
     promoter        complement(join(534..540,541..558,559..564))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    579..600
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(717..734)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(888..1476)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(968..987)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1647..2438,2439..2507))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2270..2289
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2508..2612)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2690..3145)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2827..2848)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3039..3058
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3271..3293
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3285..3302
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3286..3302
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(3309..3325,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(3309..3325,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat
       61 aatggtctca ggagttaatc caaactactg cagcctgaca gacaaatgag gatgcaaaca
      121 attttaaagt ttatctaacg ctagctgttt tgtttcttct ctctggtgca ccaacgacgg
      181 cgttttctca atcataaaga ggcttgtttt acttaaggcc aataatgttg atggatcgaa
      241 agaagagggc ttttaataaa cgagcccgtt taagctgtaa acgatgtcaa aaacatccca
      301 catcgttcag ttgaaaatag aagctctgtt tatatattgg tagagtcgac taagagattg
      361 agagaccaat atcactagtg aattcgcggc cgcctgcagg tcgaccatat gggagagctc
      421 ccaacgcgtt ggatgcatag cttgagtatt ctatagtgtc acctaaatag cttggcgtaa
      481 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
      541 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
      601 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
      661 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
      721 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
      781 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
      841 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
      901 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
      961 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     1021 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     1081 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     1141 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     1201 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     1261 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     1321 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     1381 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     1441 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     1501 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     1561 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     1621 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     1681 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     1741 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     1801 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     1861 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     1921 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     1981 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     2041 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     2101 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     2161 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     2221 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     2281 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     2341 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     2401 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     2461 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     2521 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     2581 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgat
     2641 gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcagga aattgtaagc
     2701 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     2761 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     2821 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     2881 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     2941 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     3001 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     3061 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     3121 cttaatgcgc cgctacaggg cgcgtccatt cgccattcag gctgcgcaac tgttgggaag
     3181 ggcgatcggt gcgggcctct tcgctattac gccagctggc gaaaggggga tgtgctgcaa
     3241 ggcgattaag ttgggtaacg ccagggtttt cccagtcacg acgttgtaaa acgacggcca
     3301 gtgaattgta atacgactca ctata
//
