LOCUS       sequence-252887-        2605 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  improved sgRNA backbone for tRNA-gRNA array, Position 4.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 2605)
  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 2605)
  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: pAK004
FEATURES             Location/Qualifiers
     source          1..2605
                     /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(917..1505)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(997..1016)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1683..2492)
                     /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     2406..2425
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2484..2503
                     /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 caagtttcag
      781 agctatgctg gaaacagcat agcaagttga aataaggcta gtccgttatc aacttgaaat
      841 gagaccatca ttgtcacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
      901 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
      961 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     1021 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     1081 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     1141 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     1201 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     1261 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     1321 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     1381 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     1441 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     1501 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     1561 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     1621 aaaaatgaag ttttaaatca agcccaatct gaataatgtt acaaccaatt aaccaattct
     1681 gattagaaaa actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca
     1741 ataccatatt tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc
     1801 cataggatgg caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa
     1861 cctattaatt tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg
     1921 actgaatccg gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc
     1981 cagccattac gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat
     2041 tgcgcctgag cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc
     2101 gaatgcaacc ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga
     2161 tattcttcta atacctggaa tgctgttttt ccggggatcg cagtggtgag taaccatgca
     2221 tcatcaggag tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag
     2281 tttagtctga ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga
     2341 aacaactctg gcgcatcggg cttcccatac aagcgataga ttgtcgcacc tgattgcccg
     2401 acattatcgc gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc
     2461 ggcctcgacg tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa
     2521 gcagacagtt ttattgttca tgatgatata tttttatctt gtgcaatgta acatcagaga
     2581 ttttgagaca cgggccagag ctgca
//
