LOCUS       sequence-252854-        2595 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  classic sgRNA backbone for tRNA-gRNA array, Position 4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2595)
  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 2595)
  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: pAK010
FEATURES             Location/Qualifiers
     source          1..2595
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     protein_bind    243..264
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(279..284,285..302,303..309)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    317..333
                     /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     322..344
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     341..357
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     341..357
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             353..676
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    370..426
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(427..444)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(427..443)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(436..458)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(652..671)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(907..1495)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(987..1006)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1673..2482)
                     /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     2396..2415
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2474..2493
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gacgtcattg
       61 tcgatggtct cacaacttga aaaagtggca ccgagtcggt gcaacaaagc accagtggtc
      121 tagtggtaga atagtaccct gccacggtac agacccgggt tcgattcccg gctggtgcat
      181 tgtcttcgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
      241 attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat gcttccggct
      301 cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag ctatgaccat
      361 gattacgcca agcttgcatg cctgcaggtc gactctagag gatccccggg taccgagctc
      421 gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg ttacccaact
      481 taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag aggcccgcac
      541 cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga tgcggtattt
      601 tctccttacg catctgtgcg gtatttcaca ccgcatatgg tgcactctca gtacaatctg
      661 ctctgatgcc gcatagttaa gccagccccg acacccgcca acacccgctg acgcgccctg
      721 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgacgaaga caagttttag
      781 agctagaaat agcaagttaa aataaggcta gtccgttatc aacttgaaat gagaccatca
      841 ttgtcacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
      901 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
      961 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     1021 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     1081 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     1141 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     1201 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     1261 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     1321 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     1381 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     1441 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     1501 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     1561 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     1621 ttttaaatca agcccaatct gaataatgtt acaaccaatt aaccaattct gattagaaaa
     1681 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
     1741 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
     1801 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
     1861 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
     1921 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
     1981 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     2041 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     2101 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     2161 atacctggaa tgctgttttt ccggggatcg cagtggtgag taaccatgca tcatcaggag
     2221 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     2281 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     2341 gcgcatcggg cttcccatac aagcgataga ttgtcgcacc tgattgcccg acattatcgc
     2401 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctcgacg
     2461 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     2521 ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga ttttgagaca
     2581 cgggccagag ctgca
//
