LOCUS       sequence-305768-        4403 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M1E 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 4403)
  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 4403)
  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: pDGE331
FEATURES             Location/Qualifiers
     source          1..4403
                     /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"
     CDS             complement(576..881)
                     /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     complement(678..697)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1201..1884)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     1799..1818
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1885..1987)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2182..2198)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2182..2198)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2195..2217)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2206..2222
                     /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(2230..2236,2237..2254,2255..2260))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2275..2296
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2413..2430)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2584..3172)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2664..2683)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3343..4134,4135..4203))
                     /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     3966..3985
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4204..4308)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4376..4394
                     /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 taaggtctct aagcaaaaaa aagcaccgac tcggtgccac tttttcaagt
      481 tgataacgga ctagccttat ttcaacttgc tatgctgttt ccagcatagc tctgaaacaa
      541 gtcttcgact ggctgtgtat aagggagcct gacatttata ttccccagaa catcaggtta
      601 atggcgtttt tgatgtcatt ttcgcggtgg ctgagatcag ccacttcttc cccgataacg
      661 gaaaccggca cactggccat atcggtggtc atcatgcgcc agctttcatc cccgatatgc
      721 accaccgggt aaagttcacg ggagacttta tctgacagca gacgtgcact ggccaggggg
      781 atcaccatcc gtcgcccggg cgtgtcaata atatcactct gtacatccac aaacagacga
      841 taacggctct ctcttttata ggtgtaaacc ttaaactgca tttcaccagc ccctgttctc
      901 gtcagcaaaa gagccgttca tttcaataaa ccgggcgacc tcagccatcc cttcctgatt
      961 ttccgctttc cagcgttcgg cacgcagacg acgggcttca ttctgcatgg ttgtgcttac
     1021 cagaccggag atattgacat catatatgcc ttgagcaact gatagctgtc gctgtcaact
     1081 gtcactgtaa tacgctgctt catagcatac ctctttttga catacttcgg gtatacatat
     1141 cagtatatat tcttataccg caaaaatcag cgcgcaaata cgcatactgt tatctggctt
     1201 ttagtaagcc ggatccacgc ggcgtttacg ccccccctgc cactcatcgc agtactgttg
     1261 taattcatta agcattctgc cgacatggaa gccatcacaa acggcatgat gaacctgaat
     1321 cgccagcggc atcagcacct tgtcgccttg cgtataatat ttgcccatgg tgaaaacggg
     1381 ggcgaagaag ttgtccatat tggccacgtt taaatcaaaa ctggtgaaac tcacccaggg
     1441 attggctgag acgaaaaaca tattctcaat aaacccttta gggaaatagg ccaggttttc
     1501 accgtaacac gccacatctt gcgaatatat gtgtagaaac tgccggaaat cgtcgtggta
     1561 ttcactccag agcgatgaaa acgtttcagt ttgctcatgg aaaacggtgt aacaagggtg
     1621 aacactatcc catatcacca gctcaccgtc tttcattgcc atacggaatt ccggatgagc
     1681 attcatcagg cgggcaagaa tgtgaataaa ggccggataa aacttgtgct tatttttctt
     1741 tacggtcttt aaaaaggccg taatatccag ctgaacggtc tggttatagg tacattgagc
     1801 aactgactga aatgcctcaa aatgttcttt acgatgccat tgggatatat caacggtggt
     1861 atatccagtg atttttttct ccattttagc ttccttagct cctgaaaatc tcgataactc
     1921 aaaaaatacg cccggtagtg atcttatttc attatggtga aagttggaac ctcttacgtg
     1981 ccgatcaacg tctcagaaga cttcaatcac tacttcgact ctagctgtat ataaactcag
     2041 cttcgttttc ttatctaagc gatgtgggac ttttgaagat tgttttcaac ttagtcgtga
     2101 ctgggaaaac gcaatgagac caaaatcgga tcccgggccc gtcgactgca gaggcctgca
     2161 tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     2221 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     2281 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     2341 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     2401 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     2461 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2521 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2581 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2641 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2701 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2761 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2821 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2881 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     2941 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3001 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     3061 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3121 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3181 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3241 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     3301 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     3361 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     3421 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3481 gcgagatcca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3541 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3601 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3661 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3721 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     3781 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     3841 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     3901 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     3961 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4021 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4081 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4141 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     4201 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     4261 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     4321 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     4381 gcgtatcacg aggccctttc gtc
//
