LOCUS       sequence-252856-        2599 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  classic sgRNA backbone for tRNA-gRNA array, Position 3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2599)
  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 2599)
  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: pAK009
FEATURES             Location/Qualifiers
     source          1..2599
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     protein_bind    253..274
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(289..294,295..312,313..319)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    327..343
                     /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     332..354
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     351..367
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     351..367
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             363..686
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    380..436
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(437..454)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(437..453)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(446..468)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(662..681)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(911..1499)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(991..1010)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1677..2486)
                     /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     2400..2419
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2478..2497
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gacgtcattg
       61 tcgatggtct caagtccgtt atcaacttga aaaagtggca ccgagtcggt gcaacaaagc
      121 accagtggtc tagtggtaga atagtaccct gccacggtac agacccgggt tcgattcccg
      181 gctggtgcat tgtcttcgca gctggcacga caggtttccc gactggaaag cgggcagtga
      241 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
      301 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
      361 ctatgaccat gattacgcca agcttgcatg cctgcaggtc gactctagag gatccccggg
      421 taccgagctc gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg
      481 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag
      541 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga
      601 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca
      661 gtacaatctg ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg
      721 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgacgaaga
      781 caagttttag agctagaaat agcaagttaa aataaggcta gtccgttatc aactgagacc
      841 atcattgtca catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
      901 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
      961 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     1021 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     1081 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     1141 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     1201 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     1261 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     1321 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     1381 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     1441 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     1501 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     1561 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     1621 gaagttttaa atcaagccca atctgaataa tgttacaacc aattaaccaa ttctgattag
     1681 aaaaactcat cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca
     1741 tatttttgaa aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg
     1801 atggcaagat cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt
     1861 aatttcccct cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa
     1921 tccggtgaga atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca
     1981 ttacgctcgt catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc
     2041 tgagcgagac gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc
     2101 aaccggcgca ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct
     2161 tctaatacct ggaatgctgt ttttccgggg atcgcagtgg tgagtaacca tgcatcatca
     2221 ggagtacgga taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt
     2281 ctgaccatct catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac
     2341 tctggcgcat cgggcttccc atacaagcga tagattgtcg cacctgattg cccgacatta
     2401 tcgcgagccc atttataccc atataaatca gcatccatgt tggaatttaa tcgcggcctc
     2461 gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat gtaagcagac
     2521 agttttattg ttcatgatga tatattttta tcttgtgcaa tgtaacatca gagattttga
     2581 gacacgggcc agagctgca
//
