LOCUS       sequence-261568-        2604 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  improved sgRNA backbone for tRNA-gRNA array, Position 5.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2604)
  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 2604)
  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: pAK005
FEATURES             Location/Qualifiers
     source          1..2604
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     protein_bind    237..258
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(273..278,279..296,297..303)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    311..327
                     /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     316..338
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     335..351
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     335..351
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             347..670
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    364..420
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(421..438)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(421..437)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(430..452)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(646..665)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(916..1504)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(996..1015)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1682..2491)
                     /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     2405..2424
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2483..2502
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gacgtcattg
       61 tcgatggtct cagaaaaagt ggcaccgagt cggtgcaaca aagcaccagt ggtctagtgg
      121 tagaatagta ccctgccacg gtacagaccc gggttcgatt cccggctggt gcattgtctt
      181 cgcagctggc acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat
      241 gtgagttagc tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg
      301 ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac
      361 gccaagcttg catgcctgca ggtcgactct agaggatccc cgggtaccga gctcgaattc
      421 actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg
      481 ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg
      541 cccttcccaa cagttgcgca gcctgaatgg cgaatggcgc ctgatgcggt attttctcct
      601 tacgcatctg tgcggtattt cacaccgcat atggtgcact ctcagtacaa tctgctctga
      661 tgccgcatag ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
      721 ttgtctgctc ccggcatccg cttacagaca agctgtgacg aagacaagtt tcagagctat
      781 gctggaaaca gcatagcaag ttgaaataag gctagtccgt tatcaacttg aaaaagtgtg
      841 agaccatcat tgtcacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
      901 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
      961 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     1021 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     1081 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     1141 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     1201 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     1261 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     1321 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     1381 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     1441 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     1501 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     1561 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     1621 aaaatgaagt tttaaatcaa gcccaatctg aataatgtta caaccaatta accaattctg
     1681 attagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     1741 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     1801 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     1861 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     1921 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     1981 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     2041 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     2101 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     2161 attcttctaa tacctggaat gctgtttttc cggggatcgc agtggtgagt aaccatgcat
     2221 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     2281 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     2341 acaactctgg cgcatcgggc ttcccataca agcgatagat tgtcgcacct gattgcccga
     2401 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     2461 gcctcgacgt ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag
     2521 cagacagttt tattgttcat gatgatatat ttttatcttg tgcaatgtaa catcagagat
     2581 tttgagacac gggccagagc tgca
//
