LOCUS       sequence-337780-        3502 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Insert one gRNA.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3502)
  AUTHORS   Oh Y, Lee B, Kim H, Kim SG
  TITLE     A multiplex guide RNA expression system and its efficacy for plant 
            genome engineering.
  JOURNAL   Plant Methods. 2020 Mar 12;16:37. doi: 10.1186/s13007-020-00580-x. 
            eCollection 2020.
  PUBMED    32190101
REFERENCE   2  (bases 1 to 3502)
  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: "Plant 
            Methods."; date: "2020-03-12"; volume: "16:37. doi"; pages: " 
            10.1186/s13007-020-00580-x. eCollection 2020"
COMMENT     Sequence Label: pGRNA1
FEATURES             Location/Qualifiers
     source          1..3502
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1..17
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     misc_RNA        151..226
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             686..1480
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             686..1480
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(740..759)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1350..1369
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(1498..1566,1567..2358)
                     /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     complement(1716..1735)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2529..3117
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3018..3037
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    3405..3426
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3441..3446,3447..3464,3465..3471)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3479..3495
                     /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     join(3484..3502,1..4)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 caggaaacag ctatgaccat gattacgcca agcttcacct gcgattgatt gaacaaagca
       61 ccagtggtct agtggtagaa tagtaccctg ccacggtaca gacccgggtt cgattcccgg
      121 ctggtgcaag agacctccgg agtggtctca gttttagagc tagaaatagc aagttaaaat
      181 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctaag taaggcaggt
      241 gctcgagggg cccgttatcg gggaagaagt ggctgatctc agccaccgcg aaaatgacat
      301 caaaaacgcc attaacctga tgttctgggg aatataaatg tcaggcatga gattatcaaa
      361 aaggatcttc acctagatcc ttttcacgta gaaagccagt ccgcagaaac ggtgctgacc
      421 ccggatgaat gtcagctact gggctatctg gacaagggaa aacgcaagcg caaagagaaa
      481 gcaggtagct tgcagtgggc ttacatggcg atagctagac tgggcggttt tatggacagc
      541 aagcgaaccg gaattgccag ctggggcgcc ctctggtaag gttgggaagc cctgcaaagt
      601 aaactggatg gctttcttgc cgccaaggat ctgatggcgc aggggatcaa gctctgatca
      661 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
      721 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
      781 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
      841 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
      901 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
      961 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     1021 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     1081 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     1141 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     1201 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     1261 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     1321 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     1381 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     1441 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga attgaaaaag gaagagtatg
     1501 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     1561 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     1621 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     1681 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     1741 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     1801 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     1861 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     1921 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     1981 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     2041 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     2101 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     2161 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tggttcgcgc
     2221 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     2281 acggggagcc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     2341 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     2401 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     2461 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2521 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2581 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2641 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     2701 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2761 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggattcaag acgatagtta
     2821 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     2881 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     2941 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3001 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3061 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3121 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3181 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3241 accgctcgcc gcagccgaac gaccgagcgc agcgaatcag tgagcgagga agcggaagag
     3301 cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg cagctggcac
     3361 gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt gagttagctc
     3421 actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt
     3481 gtgagcggat aacaatttca ca
//
