LOCUS       sequence-252804-        2620 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  classic sgRNA backbone for tRNA-gRNA array, Position 6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2620)
  AUTHORS   Hahn F, Korolev A, Sanjurjo Loures L, Nekrasov V
  TITLE     A modular cloning toolkit for genome editing in plants.
  JOURNAL   BMC Plant Biol. 2020 Apr 23;20(1):179. doi: 
            10.1186/s12870-020-02388-2.
  PUBMED    32326901
REFERENCE   2  (bases 1 to 2620)
  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: "BMC Plant 
            Biol."; date: "2020-04-23"; volume: "20(1):179. doi"; pages: " 
            10.1186/s12870-020-02388-2"
COMMENT     Sequence Label: pAK012
FEATURES             Location/Qualifiers
     source          1..2620
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     protein_bind    232..253
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(268..273,274..291,292..298)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    306..322
                     /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     311..333
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     330..346
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     330..346
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             342..665
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    359..415
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(416..433)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(416..432)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(425..447)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(641..660)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     misc_RNA        763..838
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     rep_origin      complement(932..1520)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1012..1031)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1698..2507)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2421..2440
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2499..2518
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gacgtcattg
       61 tcgatggtct caagtggcac cgagtcggtg caacaaagca ccagtggtct agtggtagaa
      121 tagtaccctg ccacggtaca gacccgggtt cgattcccgg ctggtgcatt gtcttcgcag
      181 ctggcacgac aggtttcccg actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag
      241 ttagctcact cattaggcac cccaggcttt acactttatg cttccggctc gtatgttgtg
      301 tggaattgtg agcggataac aatttcacac aggaaacagc tatgaccatg attacgccaa
      361 gcttgcatgc ctgcaggtcg actctagagg atccccgggt accgagctcg aattcactgg
      421 ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt aatcgccttg
      481 cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc gatcgccctt
      541 cccaacagtt gcgcagcctg aatggcgaat ggcgcctgat gcggtatttt ctccttacgc
      601 atctgtgcgg tatttcacac cgcatatggt gcactctcag tacaatctgc tctgatgccg
      661 catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
      721 tgctcccggc atccgcttac agacaagctg tgacgaagac aagttttaga gctagaaata
      781 gcaagttaaa ataaggctag tccgttatca acttgaaaaa gtggcaccga gtcggtgctt
      841 tttttttttt cgcttgagac catcattgtc acatgtgagc aaaaggccag caaaaggcca
      901 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
      961 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     1021 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     1081 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     1141 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     1201 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     1261 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     1321 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     1381 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     1441 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     1501 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     1561 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     1621 agatcctttt aaattaaaaa tgaagtttta aatcaagccc aatctgaata atgttacaac
     1681 caattaacca attctgatta gaaaaactca tcgagcatca aatgaaactg caatttattc
     1741 atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga aggagaaaac
     1801 tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat tccgactcgt
     1861 ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc aagtgagaaa
     1921 tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat ttctttccag
     1981 acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc aaccaaaccg
     2041 ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt aaaaggacaa
     2101 ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc aacaatattt
     2161 tcacctgaat caggatattc ttctaatacc tggaatgctg tttttccggg gatcgcagtg
     2221 gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg aagaggcata
     2281 aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc aacgctacct
     2341 ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaagcg atagattgtc
     2401 gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc agcatccatg
     2461 ttggaattta atcgcggcct cgacgtttcc cgttgaatat ggctcataac accccttgta
     2521 ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt atcttgtgca
     2581 atgtaacatc agagattttg agacacgggc cagagctgca
//
