LOCUS       sequence-337781-        3501 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 3501)
  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 3501)
  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: pGRNA2
FEATURES             Location/Qualifiers
     source          1..3501
                     /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        150..225
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             685..1479
                     /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             685..1479
                     /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(739..758)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1349..1368
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(1497..1565,1566..2357)
                     /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(1715..1734)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2528..3116
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3017..3036
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    3404..3425
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3440..3445,3446..3463,3464..3470)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3478..3494
                     /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(3483..3501,1..4)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
ORIGIN
        1 caggaaacag ctatgaccat gattacgcca agcttcacct gctaagtaag aacaaagcac
       61 cagtggtcta gtggtagaat agtaccctgc cacggtacag acccgggttc gattcccggc
      121 tggtgcaaga gacctccgga gtggtctcag ttttagagct agaaatagca agttaaaata
      181 aggctagtcc gttatcaact tgaaaaagtg gcaccgagtc ggtgcggctg gctgcaggtg
      241 ctcgaggggc ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc
      301 aaaaacgcca ttaacctgat gttctgggga atataaatgt caggcatgag attatcaaaa
      361 aggatcttca cctagatcct tttcacgtag aaagccagtc cgcagaaacg gtgctgaccc
      421 cggatgaatg tcagctactg ggctatctgg acaagggaaa acgcaagcgc aaagagaaag
      481 caggtagctt gcagtgggct tacatggcga tagctagact gggcggtttt atggacagca
      541 agcgaaccgg aattgccagc tggggcgccc tctggtaagg ttgggaagcc ctgcaaagta
      601 aactggatgg ctttcttgcc gccaaggatc tgatggcgca ggggatcaag ctctgatcaa
      661 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
      721 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
      781 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
      841 ctgtccggtg ccctgaatga actgcaagac gaggcagcgc ggctatcgtg gctggccacg
      901 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
      961 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     1021 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     1081 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     1141 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     1201 aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     1261 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     1321 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     1381 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     1441 cgcatcgcct tctatcgcct tcttgacgag ttcttctgaa ttgaaaaagg aagagtatga
     1501 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     1561 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     1621 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     1681 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     1741 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     1801 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     1861 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     1921 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     1981 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     2041 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     2101 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     2161 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt ggttcgcgcg
     2221 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     2281 cggggagcca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     2341 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     2401 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     2461 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     2521 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     2581 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     2641 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     2701 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     2761 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggattcaaga cgatagttac
     2821 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     2881 gaacgaccta caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc
     2941 ccgaagggag aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca
     3001 cgagggagct tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc
     3061 tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg
     3121 ccagcaacgc ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgttct
     3181 ttcctgcgtt atcccctgat tctgtggata accgtattac cgcctttgag tgagctgata
     3241 ccgctcgccg cagccgaacg accgagcgca gcgaatcagt gagcgaggaa gcggaagagc
     3301 gcccaatacg caaaccgcct ctccccgcgc gttggccgat tcattaatgc agctggcacg
     3361 acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc aattaatgtg agttagctca
     3421 ctcattaggc accccaggct ttacacttta tgcttccggc tcgtatgttg tgtggaattg
     3481 tgagcggata acaatttcac a
//
