LOCUS       sequence-337784-        3425 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 3425)
  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 3425)
  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: pGRNA2e
FEATURES             Location/Qualifiers
     source          1..3425
                     /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"
     CDS             609..1403
                     /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             609..1403
                     /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(663..682)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     1273..1292
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(1421..1489,1490..2281)
                     /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(1639..1658)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2452..3040
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2941..2960
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     protein_bind    3328..3349
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3364..3369,3370..3387,3388..3394)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3402..3418
                     /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(3407..3425,1..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 tttgaaagca ggtgctcgag gggcccgtta
      181 tcggggaaga agtggctgat ctcagccacc gcgaaaatga catcaaaaac gccattaacc
      241 tgatgttctg gggaatataa atgtcaggca tgagattatc aaaaaggatc ttcacctaga
      301 tccttttcac gtagaaagcc agtccgcaga aacggtgctg accccggatg aatgtcagct
      361 actgggctat ctggacaagg gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg
      421 ggcttacatg gcgatagcta gactgggcgg ttttatggac agcaagcgaa ccggaattgc
      481 cagctggggc gccctctggt aaggttggga agccctgcaa agtaaactgg atggctttct
      541 tgccgccaag gatctgatgg cgcaggggat caagctctga tcaagagaca ggatgaggat
      601 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
      661 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
      721 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
      781 atgaactgca agacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
      841 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
      901 cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc atcatggctg
      961 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     1021 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     1081 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgagca
     1141 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     1201 tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg gcggaccgct
     1261 atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc gaatgggctg
     1321 accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc gccttctatc
     1381 gccttcttga cgagttcttc tgaattgaaa aaggaagagt atgagtattc aacatttccg
     1441 tgtcgccctt attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac
     1501 gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact
     1561 ggatctcaac agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat
     1621 gagcactttt aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga
     1681 gcaactcggt cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac
     1741 agaaaagcat cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat
     1801 gagtgataac actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac
     1861 cgcttttttg cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct
     1921 gaatgaagcc ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac
     1981 gttgcgcaaa ctattaactg gcgaactact tactctagct tcccggcaac aattaataga
     2041 ctggatggag gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg
     2101 gtttattgct gataaatctg gagccggtga gcgtggttcg cgcggtatca ttgcagcact
     2161 ggggccagat ggtaagccct cccgtatcgt agttatctac acgacgggga gccaggcaac
     2221 tatggatgaa cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta
     2281 actgtcagac caagtttact catatatact ttagattgat ttaaaacttc atttttaatt
     2341 taaaaggatc taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga
     2401 gttttcgttc cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc
     2461 tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt
     2521 ttgtttgccg gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc
     2581 gcagatacca aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc
     2641 tgtagcaccg cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg
     2701 cgataagtcg tgtcttaccg ggttggattc aagacgatag ttaccggata aggcgcagcg
     2761 gtcgggctga acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga
     2821 actgagatac ctacagcgtg agctatgaga aagcgccacg cttcccgaag ggagaaaggc
     2881 ggacaggtat ccggtaagcg gcagggtcgg aacaggagag cgcacgaggg agcttccagg
     2941 gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc cacctctgac ttgagcgtcg
     3001 atttttgtga tgctcgtcag gggggcggag cctatggaaa aacgccagca acgcggcctt
     3061 tttacggttc ctggcctttt gctggccttt tgctcacatg ttctttcctg cgttatcccc
     3121 tgattctgtg gataaccgta ttaccgcctt tgagtgagct gataccgctc gccgcagccg
     3181 aacgaccgag cgcagcgaat cagtgagcga ggaagcggaa gagcgcccaa tacgcaaacc
     3241 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
     3301 gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca
     3361 ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt
     3421 tcaca
//
