LOCUS       sequence-305925-        4443 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M4E 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 4443)
  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 4443)
  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: pDGE660
FEATURES             Location/Qualifiers
     source          1..4443
                     /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        608..710
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             711..1394
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(777..796)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1714..2019
                     /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     1898..1917
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2222..2238)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2222..2238)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2235..2257)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2246..2262
                     /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(2270..2276,2277..2294,2295..2300))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2315..2336
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2453..2470)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2624..3212)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2704..2723)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3383..4174,4175..4243))
                     /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     4006..4025
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4244..4348)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4416..4434
                     /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 ttggtctcaa tggcaagaga aataagaaaa aggccggccc atttagattg
      481 aagatccagg cccaggcccg taaaagaaac ccaacaagca aattctccct catcgcttat
      541 acaaagctac tttgcctcgt ttatatagcg gaatatgaac atgtatgatt gaagtcttct
      601 gagacgttga tcggcacgta agaggttcca actttcacca taatgaaata agatcactac
      661 cgggcgtatt ttttgagtta tcgagatttt caggagctaa ggaagctaaa atggagaaaa
      721 aaatcactgg atataccacc gttgatatat cccaatggca tcgtaaagaa cattttgagg
      781 catttcagtc agttgctcaa tgtacctata accagaccgt tcagctggat attacggcct
      841 ttttaaagac cgtaaagaaa aataagcaca agttttatcc ggcctttatt cacattcttg
      901 cccgcctgat gaatgctcat ccggaattcc gtatggcaat gaaagacggt gagctggtga
      961 tatgggatag tgttcaccct tgttacaccg ttttccatga gcaaactgaa acgttttcat
     1021 cgctctggag tgaataccac gacgatttcc ggcagtttct acacatatat tcgcaagatg
     1081 tggcgtgtta cggtgaaaac ctggcctatt tccctaaagg gtttattgag aatatgtttt
     1141 tcgtctcagc caatccctgg gtgagtttca ccagttttga tttaaacgtg gccaatatgg
     1201 acaacttctt cgcccccgtt ttcaccatgg gcaaatatta tacgcaaggc gacaaggtgc
     1261 tgatgccgct ggcgattcag gttcatcatg ccgtttgtga tggcttccat gtcggcagaa
     1321 tgcttaatga attacaacag tactgcgatg agtggcaggg ggggcgtaaa cgccgcgtgg
     1381 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat
     1441 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag
     1501 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca
     1561 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac
     1621 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg
     1681 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa
     1741 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
     1801 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa
     1861 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     1921 agtgtgccgg tttccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     1981 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac
     2041 agccagtcga agacttgttt cagagctatg ctggaaacag catagcaagt tgaaataagg
     2101 ctagtccgtt atcaacttga aaaagtggca ccgagtcggt gctttttttt gcttagagac
     2161 ctttatcgga tcccgggccc gtcgactgca gaggcctgca tgcaagcttg gcgtaatcat
     2221 ggtcatagct gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag
     2281 ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg
     2341 cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa
     2401 tcggccaacg cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca
     2461 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     2521 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     2581 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     2641 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     2701 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     2761 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     2821 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     2881 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     2941 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     3001 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     3061 gaagaacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     3121 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     3181 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     3241 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     3301 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     3361 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     3421 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     3481 gggagggctt accatctggc cccagtgctg caatgatacc gcgagatcca cgctcaccgg
     3541 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     3601 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     3661 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     3721 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     3781 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     3841 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     3901 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     3961 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     4021 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     4081 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     4141 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     4201 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     4261 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     4321 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct
     4381 aagaaaccat tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc
     4441 gtc
//
