LOCUS       sequence-305781-        4387 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M2 shuttle vector containing the Arabidopsis thaliana U6 promoter 
            for preparation of single guide RNA transcriptional units by cloning
            of hybridized oligonucleotides..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4387)
  AUTHORS   Stuttmann J, Barthel K, Martin P, Ordon J, Erickson JL, Herr R, 
            Ferik F, Kretschmer C, Berner T, Keilwagen J, Marillonnet S, Bonas U
  TITLE     Highly efficient multiplex editing: One-shot generation of 8x 
            Nicotiana benthamiana and 12x Arabidopsis mutants.
  JOURNAL   Plant J. 2021 Feb 12. doi: 10.1111/tpj.15197.
  PUBMED    33577114
REFERENCE   2  (bases 1 to 4387)
  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 J. 
            2021 Feb 12. doi: 10.1111/tpj.15197."
COMMENT     Sequence Label: pDGE333
FEATURES             Location/Qualifiers
     source          1..4387
                     /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"
     promoter        552..654
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             655..1338
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(721..740)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1658..1963
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1842..1861
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2166..2182)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2166..2182)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2179..2201)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2190..2206
                     /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(2214..2220,2221..2238,2239..2244))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2259..2280
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2397..2414)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2568..3156)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2648..2667)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3327..4118,4119..4187))
                     /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     3950..3969
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4188..4292)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4360..4378
                     /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 caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acctcgcgaa
      421 tgcatctaga ttttggtctc acccttaagt tgaaaacaat cttcaaaagt cccacatcgc
      481 ttagataaga aaacgaagct gagtttatat acagctagag tcgaagtagt gattgaagtc
      541 ttctgagacg ttgatcggca cgtaagaggt tccaactttc accataatga aataagatca
      601 ctaccgggcg tattttttga gttatcgaga ttttcaggag ctaaggaagc taaaatggag
      661 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
      721 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
      781 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
      841 cttgcccgcc tgatgaatgc tcatccggaa ttccgtatgg caatgaaaga cggtgagctg
      901 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
      961 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
     1021 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
     1081 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
     1141 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
     1201 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc
     1261 agaatgctta atgaattaca acagtactgc gatgagtggc agggggggcg taaacgccgc
     1321 gtggatccgg cttactaaaa gccagataac agtatgcgta tttgcgcgct gatttttgcg
     1381 gtataagaat atatactgat atgtataccc gaagtatgtc aaaaagaggt atgctatgaa
     1441 gcagcgtatt acagtgacag ttgacagcga cagctatcag ttgctcaagg catatatgat
     1501 gtcaatatct ccggtctggt aagcacaacc atgcagaatg aagcccgtcg tctgcgtgcc
     1561 gaacgctgga aagcggaaaa tcaggaaggg atggctgagg tcgcccggtt tattgaaatg
     1621 aacggctctt ttgctgacga gaacaggggc tggtgaaatg cagtttaagg tttacaccta
     1681 taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta ttgacacgcc
     1741 cgggcgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata aagtctcccg
     1801 tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga ccaccgatat
     1861 ggccagtgtg ccggtttccg ttatcgggga agaagtggct gatctcagcc accgcgaaaa
     1921 tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag gctcccttat
     1981 acacagccag tcgaagactt gtttcagagc tatgctggaa acagcatagc aagttgaaat
     2041 aaggctagtc cgttatcaac ttgaaaaagt ggcaccgagt cggtgctttt ttttcggttg
     2101 agaccaaaat cggatcccgg gcccgtcgac tgcagaggcc tgcatgcaag cttggcgtaa
     2161 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     2221 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     2281 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     2341 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     2401 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     2461 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     2521 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     2581 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     2641 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     2701 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     2761 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     2821 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     2881 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     2941 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     3001 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     3061 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     3121 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     3181 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     3241 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     3301 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     3361 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     3421 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga tccacgctca
     3481 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     3541 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     3601 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     3661 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     3721 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     3781 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     3841 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     3901 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     3961 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     4021 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     4081 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     4141 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     4201 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     4261 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     4321 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
     4381 tttcgtc
//
