LOCUS       sequence-305772-        4402 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  M1 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 4402)
  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 4402)
  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: pDGE332
FEATURES             Location/Qualifiers
     source          1..4402
                     /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(577..882)
                     /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(679..698)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1202..1885)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     1800..1819
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(1886..1988)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2181..2197)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2181..2197)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2194..2216)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2205..2221
                     /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(2229..2235,2236..2253,2254..2259))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2274..2295
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2412..2429)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2583..3171)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2663..2682)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3342..4133,4134..4202))
                     /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     3965..3984
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4203..4307)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4375..4393
                     /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 aagggaaaaa aaagcaccga ctcggtgcca ctttttcaag
      481 ttgataacgg actagcctta tttcaacttg ctatgctgtt tccagcatag ctctgaaaca
      541 agtcttcgac tggctgtgta taagggagcc tgacatttat attccccaga acatcaggtt
      601 aatggcgttt ttgatgtcat tttcgcggtg gctgagatca gccacttctt ccccgataac
      661 ggaaaccggc acactggcca tatcggtggt catcatgcgc cagctttcat ccccgatatg
      721 caccaccggg taaagttcac gggagacttt atctgacagc agacgtgcac tggccagggg
      781 gatcaccatc cgtcgcccgg gcgtgtcaat aatatcactc tgtacatcca caaacagacg
      841 ataacggctc tctcttttat aggtgtaaac cttaaactgc atttcaccag cccctgttct
      901 cgtcagcaaa agagccgttc atttcaataa accgggcgac ctcagccatc ccttcctgat
      961 tttccgcttt ccagcgttcg gcacgcagac gacgggcttc attctgcatg gttgtgctta
     1021 ccagaccgga gatattgaca tcatatatgc cttgagcaac tgatagctgt cgctgtcaac
     1081 tgtcactgta atacgctgct tcatagcata cctctttttg acatacttcg ggtatacata
     1141 tcagtatata ttcttatacc gcaaaaatca gcgcgcaaat acgcatactg ttatctggct
     1201 tttagtaagc cggatccacg cggcgtttac gccccccctg ccactcatcg cagtactgtt
     1261 gtaattcatt aagcattctg ccgacatgga agccatcaca aacggcatga tgaacctgaa
     1321 tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg
     1381 gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg
     1441 gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt
     1501 caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt
     1561 attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt
     1621 gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaat tccggatgag
     1681 cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct
     1741 ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag
     1801 caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg
     1861 tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact
     1921 caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt
     1981 gccgatcaac gtctcagaag acttcaatca ctacttcgac tctagctgta tataaactca
     2041 gcttcgtttt cttatctaag cgatgtggga cttttgaaga ttgttttcaa cttagtcgtg
     2101 actgggaaaa cgcaatgaga ccaatcggat cccgggcccg tcgactgcag aggcctgcat
     2161 gcaagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     2221 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     2281 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     2341 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     2401 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     2461 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     2521 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     2581 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     2641 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     2701 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     2761 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     2821 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     2881 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     2941 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     3001 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     3061 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     3121 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     3181 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     3241 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     3301 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     3361 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     3421 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     3481 cgagatccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     3541 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     3601 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     3661 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     3721 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     3781 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     3841 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     3901 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     3961 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     4021 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     4081 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     4141 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     4201 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     4261 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     4321 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     4381 cgtatcacga ggccctttcg tc
//
