LOCUS       sequence-305930-        4463 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M5 shuttle vector containing the Solanum lycopersicum 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 4463)
  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 4463)
  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: pDGE661
FEATURES             Location/Qualifiers
     source          1..4463
                     /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        630..732
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             733..1416
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(799..818)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1736..2041
                     /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     1920..1939
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2242..2258)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2242..2258)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2255..2277)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2266..2282
                     /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(2290..2296,2297..2314,2315..2320))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2335..2356
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2473..2490)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2644..3232)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2724..2743)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3403..4194,4195..4263))
                     /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     4026..4045
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4264..4368)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4436..4454
                     /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 tttggtctca gactgccagc ttctatgagt actgacaaga gaaataagaa
      481 aaaggccggc ccatttagat tgaagatcca ggcccaggcc cgtaaaagaa acccaacaag
      541 caaattctcc ctcatcgctt atacaaagct actttgcctc gtttatatag cggaatatga
      601 acatgtatga ttgaagtctt ctgagacgtt gatcggcacg taagaggttc caactttcac
      661 cataatgaaa taagatcact accgggcgta ttttttgagt tatcgagatt ttcaggagct
      721 aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat atcccaatgg
      781 catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta taaccagacc
      841 gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca caagttttat
      901 ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt ccgtatggca
      961 atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac cgttttccat
     1021 gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt ccggcagttt
     1081 ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta tttccctaaa
     1141 gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt caccagtttt
     1201 gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat gggcaaatat
     1261 tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca tgccgtttgt
     1321 gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga tgagtggcag
     1381 ggggggcgta aacgccgcgt ggatccggct tactaaaagc cagataacag tatgcgtatt
     1441 tgcgcgctga tttttgcggt ataagaatat atactgatat gtatacccga agtatgtcaa
     1501 aaagaggtat gctatgaagc agcgtattac agtgacagtt gacagcgaca gctatcagtt
     1561 gctcaaggca tatatgatgt caatatctcc ggtctggtaa gcacaaccat gcagaatgaa
     1621 gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc aggaagggat ggctgaggtc
     1681 gcccggttta ttgaaatgaa cggctctttt gctgacgaga acaggggctg gtgaaatgca
     1741 gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg atgtacagag
     1801 tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg cacgtctgct
     1861 gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg aaagctggcg
     1921 catgatgacc accgatatgg ccagtgtgcc ggtttccgtt atcggggaag aagtggctga
     1981 tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct ggggaatata
     2041 aatgtcaggc tcccttatac acagccagtc gaagacttgt ttcagagcta tgctggaaac
     2101 agcatagcaa gttgaaataa ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg
     2161 gtgctttttt tttgcgtgag accaatcgga tcccgggccc gtcgactgca gaggcctgca
     2221 tgcaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     2281 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     2341 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     2401 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     2461 ctcttccgct tcctcgctca ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt
     2521 atcagctcac tcaaaggcgg taatacggtt atccacagaa tcaggggata acgcaggaaa
     2581 gaacatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     2641 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     2701 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     2761 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     2821 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     2881 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     2941 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     3001 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     3061 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     3121 taccttcgga aaaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     3181 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     3241 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     3301 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     3361 taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat gcttaatcag
     3421 tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct gactccccgt
     3481 cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg caatgatacc
     3541 gcgagatcca cgctcaccgg ctccagattt atcagcaata aaccagccag ccggaagggc
     3601 cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta attgttgccg
     3661 ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg ccattgctac
     3721 aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg gttcccaacg
     3781 atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc
     3841 tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta tggcagcact
     3901 gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg gtgagtactc
     3961 aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat
     4021 acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg gaaaacgttc
     4081 ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga tgtaacccac
     4141 tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg ggtgagcaaa
     4201 aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat gttgaatact
     4261 catactcttc ctttttcaat attattgaag catttatcag ggttattgtc tcatgagcgg
     4321 atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca catttccccg
     4381 aaaagtgcca cctgacgtct aagaaaccat tattatcatg acattaacct ataaaaatag
     4441 gcgtatcacg aggccctttc gtc
//
