LOCUS       sequence-496204-        3127 bp    DNA     circular SYN 06-JAN-2026
DEFINITION  gRNA insert vector for Golden Gate Assembly, 3 gene or 6 gRNAs 
            version.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3127)
  AUTHORS   Kozgunova E
  TITLE     tRNA-Based Polycistronic CRISPR/Cas9 System Boosts Efficiency of 
            Multi-Gene Deletion in the Moss Physcomitrella
  JOURNAL   bioRxiv 2025.07.29.667574
REFERENCE   2  (bases 1 to 3127)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 6, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            2025.07.29.667574"
COMMENT     Sequence Label: EKv414(Gold Cas-guide insert6 3 gene version)
FEATURES             Location/Qualifiers
     source          1..3127
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_RNA        770..845
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(906..922)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(906..922)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(919..941)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    930..946
                     /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)."
     promoter        complement(join(954..960,961..978,979..984))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    999..1020
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1137..1154)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1308..1896)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1388..1407)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2067..2858,2859..2927))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     2690..2709
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2928..3032)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3100..3118
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcac aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa gcttggtctc atcagtcgat
      421 tgaatgtcca ttgaagcaga cgtgttgcga caggttagcg acgatgggtg tagatgtgat
      481 gtgatgtgat ggtgtggttc ttccacggcg gcgtccttgc ggtggcggag aaggggatat
      541 cccgaaggag cggcagcggg agagcacaag cagaaagggt gcagtgagtg agtgggtcca
      601 gctgggtggc tggccgagtg gacgcgaccg ggtttcgagg ggggggggga gaaaagggat
      661 ggagcgaggg atataaccca catggaatgg aggtgggtgt gaaggcgggt atataggaag
      721 gtggaggact tacaacccat gaggtcttcg ccccatggta tgaagactcg ttttagagct
      781 agaaatagca agttaaaata aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc
      841 ggtgcttttt ttccttagag acctctagag gatccccggg taccgagctc gaattcgtaa
      901 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
      961 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     1021 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     1081 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     1141 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     1201 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     1261 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccatgggctc
     1321 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     1381 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     1441 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     1501 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     1561 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     1621 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     1681 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     1741 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     1801 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     1861 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     1921 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     1981 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     2041 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     2101 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     2161 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga accacgctca
     2221 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     2281 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     2341 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     2401 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     2461 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     2521 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     2581 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     2641 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     2701 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     2761 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     2821 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     2881 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     2941 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     3001 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     3061 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     3121 tttcgtc
//
