LOCUS       sequence-252896-        2609 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  improved sgRNA backbone for tRNA-gRNA array, Position 2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2609)
  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 2609)
  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: pAK002
FEATURES             Location/Qualifiers
     source          1..2609
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     protein_bind    263..284
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(299..304,305..322,323..329)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    337..353
                     /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     342..364
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     361..377
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     361..377
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             373..696
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    390..446
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(447..464)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(447..463)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(456..478)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(672..691)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(921..1509)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1001..1020)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1687..2496)
                     /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     2410..2429
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2488..2507
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gacgtcattg
       61 tcgatggtct caaaataagg ctagtccgtt atcaacttga aaaagtggca ccgagtcggt
      121 gcaacaaagc accagtggtc tagtggtaga atagtaccct gccacggtac agacccgggt
      181 tcgattcccg gctggtgcat tgtcttcgca gctggcacga caggtttccc gactggaaag
      241 cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt
      301 tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca
      361 caggaaacag ctatgaccat gattacgcca agcttgcatg cctgcaggtc gactctagag
      421 gatccccggg taccgagctc gaattcactg gccgtcgttt tacaacgtcg tgactgggaa
      481 aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt
      541 aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa
      601 tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg
      661 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca
      721 acacccgctg acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct
      781 gtgacgaaga caagtttcag agctatgctg gaaacagcat agcaagttga aataaggcta
      841 gtctgagacc atcattgtca catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
      901 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
      961 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     1021 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     1081 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     1141 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     1201 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     1261 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     1321 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     1381 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     1441 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     1501 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     1561 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     1621 aattaaaaat gaagttttaa atcaagccca atctgaataa tgttacaacc aattaaccaa
     1681 ttctgattag aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt
     1741 atcaatacca tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca
     1801 gttccatagg atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat
     1861 acaacctatt aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt
     1921 gacgactgaa tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac
     1981 aggccagcca ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg
     2041 tgattgcgcc tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg
     2101 aatcgaatgc aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc
     2161 aggatattct tctaatacct ggaatgctgt ttttccgggg atcgcagtgg tgagtaacca
     2221 tgcatcatca ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag
     2281 ccagtttagt ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt
     2341 cagaaacaac tctggcgcat cgggcttccc atacaagcga tagattgtcg cacctgattg
     2401 cccgacatta tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa
     2461 tcgcggcctc gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     2521 gtaagcagac agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca
     2581 gagattttga gacacgggcc agagctgca
//
