LOCUS       sequence-305804-        4384 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M4E 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 4384)
  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 4384)
  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: pDGE337
FEATURES             Location/Qualifiers
     source          1..4384
                     /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        550..652
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             653..1336
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(719..738)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1656..1961
                     /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     1840..1859
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     primer_bind     complement(2163..2179)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2163..2179)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2176..2198)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2187..2203
                     /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(2211..2217,2218..2235,2236..2241))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2256..2277
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2394..2411)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2565..3153)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2645..2664)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3324..4115,4116..4184))
                     /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     3947..3966
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4185..4289)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4357..4375
                     /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 atggtaattg aaaacaatct tcaaaagtcc cacatcgctt
      481 agataagaaa acgaagctga gtttatatac agctagagtc gaagtagtga ttgaagtctt
      541 ctgagacgtt gatcggcacg taagaggttc caactttcac cataatgaaa taagatcact
      601 accgggcgta ttttttgagt tatcgagatt ttcaggagct aaggaagcta aaatggagaa
      661 aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga
      721 ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc
      781 ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct
      841 tgcccgcctg atgaatgctc atccggaatt ccgtatggca atgaaagacg gtgagctggt
      901 gatatgggat agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc
      961 atcgctctgg agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga
     1021 tgtggcgtgt tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt
     1081 tttcgtctca gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat
     1141 ggacaacttc ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt
     1201 gctgatgccg ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag
     1261 aatgcttaat gaattacaac agtactgcga tgagtggcag ggggggcgta aacgccgcgt
     1321 ggatccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt
     1381 ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgaagc
     1441 agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt
     1501 caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga
     1561 acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa
     1621 cggctctttt gctgacgaga acaggggctg gtgaaatgca gtttaaggtt tacacctata
     1681 aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg
     1741 ggcgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg
     1801 aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg
     1861 ccagtgtgcc ggtttccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg
     1921 acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac
     1981 acagccagtc gaagacttgt ttcagagcta tgctggaaac agcatagcaa gttgaaataa
     2041 ggctagtccg ttatcaactt gaaaaagtgg caccgagtcg gtgctttttt ttgcttagag
     2101 accttatcgg atcccgggcc cgtcgactgc agaggcctgc atgcaagctt ggcgtaatca
     2161 tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca caacatacga
     2221 gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact cacattaatt
     2281 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     2341 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     2401 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     2461 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     2521 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     2581 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     2641 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     2701 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     2761 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     2821 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     2881 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     2941 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     3001 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     3061 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     3121 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     3181 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     3241 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     3301 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     3361 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     3421 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagatcc acgctcaccg
     3481 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     3541 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     3601 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     3661 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     3721 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     3781 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     3841 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     3901 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     3961 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     4021 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     4081 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     4141 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     4201 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     4261 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctgacgtc
     4321 taagaaacca ttattatcat gacattaacc tataaaaata ggcgtatcac gaggcccttt
     4381 cgtc
//
