LOCUS       sequence-252877-        2630 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  improved sgRNA backbone for tRNA-gRNA array, Position 6.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2630)
  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 2630)
  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: pAK006
FEATURES             Location/Qualifiers
     source          1..2630
                     /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"
     rep_origin      complement(942..1530)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1022..1041)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1708..2517)
                     /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     2431..2450
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2509..2528
                     /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 aagtttcaga gctatgctgg
      781 aaacagcata gcaagttgaa ataaggctag tccgttatca acttgaaaaa gtggcaccga
      841 gtcggtgctt tttttttttt cgcttgagac catcattgtc acatgtgagc aaaaggccag
      901 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
      961 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     1021 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     1081 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     1141 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     1201 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     1261 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     1321 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     1381 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     1441 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     1501 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     1561 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     1621 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaagccc aatctgaata
     1681 atgttacaac caattaacca attctgatta gaaaaactca tcgagcatca aatgaaactg
     1741 caatttattc atatcaggat tatcaatacc atatttttga aaaagccgtt tctgtaatga
     1801 aggagaaaac tcaccgaggc agttccatag gatggcaaga tcctggtatc ggtctgcgat
     1861 tccgactcgt ccaacatcaa tacaacctat taatttcccc tcgtcaaaaa taaggttatc
     1921 aagtgagaaa tcaccatgag tgacgactga atccggtgag aatggcaaaa gtttatgcat
     1981 ttctttccag acttgttcaa caggccagcc attacgctcg tcatcaaaat cactcgcatc
     2041 aaccaaaccg ttattcattc gtgattgcgc ctgagcgaga cgaaatacgc gatcgctgtt
     2101 aaaaggacaa ttacaaacag gaatcgaatg caaccggcgc aggaacactg ccagcgcatc
     2161 aacaatattt tcacctgaat caggatattc ttctaatacc tggaatgctg tttttccggg
     2221 gatcgcagtg gtgagtaacc atgcatcatc aggagtacgg ataaaatgct tgatggtcgg
     2281 aagaggcata aattccgtca gccagtttag tctgaccatc tcatctgtaa catcattggc
     2341 aacgctacct ttgccatgtt tcagaaacaa ctctggcgca tcgggcttcc catacaagcg
     2401 atagattgtc gcacctgatt gcccgacatt atcgcgagcc catttatacc catataaatc
     2461 agcatccatg ttggaattta atcgcggcct cgacgtttcc cgttgaatat ggctcataac
     2521 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgatg atatattttt
     2581 atcttgtgca atgtaacatc agagattttg agacacgggc cagagctgca
//
