LOCUS       sequence-316585-        3465 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AtpU6-26 Golden Gate level 0 piece to express gRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3465)
  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 3465)
  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-AtpU6-26
FEATURES             Location/Qualifiers
     source          1..3465
                     /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(590..608)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(591..608)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(626..642)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(626..642)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(639..661)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    650..666
                     /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(674..680,681..698,699..704))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    719..740
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(857..874)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1028..1616)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1108..1127)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1787..2578,2579..2647))
                     /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     2410..2429
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2648..2752)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2830..3285)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2967..2988)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3179..3198
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3411..3433
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3425..3442
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3426..3442
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     join(3449..3465,1..3)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        join(3449..3465,1..2)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gggcgaattg ggcccgacgt cgcatgctcc cggccgccat ggcggccgcg ggaattcgat
       61 tttggtctct caatcactac ttcgactcta gctgtatata aactcagctt cgttttctta
      121 tctaagcgat gtgggacttt tgaagattgt tttcaactta aatgggccta tataagaaat
      181 actattgttc tttcccatat aaatgggcct gcttctcttc tttcagattc ccaggggcct
      241 tttgaagatt atcttcatat cttaagaatg aagatgtttt attcaatcaa attcttgaag
      301 gttcgatgcc taatcattct aatcctggga caaactatga aacaagatac aaaaactccg
      361 aatggaaagt taaaaagaag aaaacgaaag ctacggttca agaaaatgta agctgataaa
      421 caaaaaaaaa ctgtatgaac gaagaagaag aaaaaaagct aagaagaaat gatgtattgt
      481 gcggaaggca agtcgctcct gagaccatta atcactagtg aattcgcggc cgcctgcagg
      541 tcgaccatat gggagagctc ccaacgcgtt ggatgcatag cttgagtatt ctatagtgtc
      601 acctaaatag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
      661 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
      721 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
      781 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
      841 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
      901 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
      961 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     1021 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     1081 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     1141 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     1201 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     1261 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     1321 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     1381 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     1441 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     1501 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     1561 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     1621 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     1681 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     1741 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     1801 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     1861 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     1921 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     1981 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     2041 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     2101 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     2161 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     2221 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     2281 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     2341 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     2401 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     2461 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     2521 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     2581 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     2641 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     2701 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     2761 cccgaaaagt gccacctgat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac
     2821 cgcatcagga aattgtaagc gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa
     2881 tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa tcaaaagaat
     2941 agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta ttaaagaacg
     3001 tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac
     3061 catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat cggaacccta
     3121 aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg agaaaggaag
     3181 ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg
     3241 taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtccatt cgccattcag
     3301 gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac gccagctggc
     3361 gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt cccagtcacg
     3421 acgttgtaaa acgacggcca gtgaattgta atacgactca ctata
//
