LOCUS       sequence-148626-        4492 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA shuttle vector; module 1.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4492)
  AUTHORS   Ordon J, Gantner J, Kemna J, Schwalgun L, Reschke M, Streubel J, 
            Boch J, Stuttmann J
  TITLE     Generation of chromosomal deletions in dicotyledonous plants 
            employing a user-friendly genome editing toolkit.
  JOURNAL   Plant J. 2016 Aug 31. doi: 10.1111/tpj.13319.
  PUBMED    27579989
REFERENCE   2  (bases 1 to 4492)
  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. 
            2016 Aug 31. doi: 10.1111/tpj.13319."
COMMENT     Sequence Label: pDGE5
FEATURES             Location/Qualifiers
     source          1..4492
                     /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        670..772
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             773..1432
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(839..858)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1774..2079
                     /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     1958..1977
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     misc_RNA        2117..2192
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(2271..2287)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2271..2287)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2284..2306)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2295..2311
                     /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(2319..2325,2326..2343,2344..2349))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2364..2385
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2502..2519)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2673..3261)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2753..2772)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3432..4223,4224..4292))
                     /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     4055..4074
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4293..4397)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4465..4483
                     /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 ttggtctcat tgcgaatatc atccggtgca gcagctgctg cagcaattaa
      481 acttgcacag aggccagaaa acgctgggaa gctatttgtg gcgatattcc cgagtttcgg
      541 ggagaggtat ctatcaacgg tactaagttg aaaacaatct tcaaaagtcc cacatcgctt
      601 agataagaaa acgaagctga gtttatatac agctagagtc gaagtagtga ttgaagtctt
      661 ctgagacgtt gatcggcacg taagaggttc caactttcac cataatgaaa taagatcact
      721 accgggcgta ttttttgagt tatcgagatt ttcaggagct aaggaagcta aaatggagaa
      781 aaaaatcact ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga
      841 ggcatttcag tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc
      901 ctttttaaag accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct
      961 tgcccgcctg atgaatgctc atccggaatt ccgtatggca atgaaagacg gtgagctggt
     1021 gatatgggat agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc
     1081 atcgctctgg agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga
     1141 tgtggcgtgt tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt
     1201 tttcgtctca gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat
     1261 ggacaacttc ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt
     1321 gctgatgccg ctggcgattc aggttcatca tgccgtctgt gatggcttcc atgtcggcag
     1381 aatgcttaat gaattacaac agtactgcga tgagtggcag ggcggggcgt aatcgcgtgg
     1441 atccggctta ctaaaagcca gataacagta tgcgtatttg cgcgctgatt tttgcggtat
     1501 aagaatatat actgatatgt atacccgaag tatgtcaaaa agaggtatgc tatgaagcag
     1561 cgtattacag tgacagttga cagcgacagc tatcagttgc tcaaggcata tatgatgtca
     1621 atatctccgg tctggtaagc acaaccatgc agaatgaagc ccgtcgtctg cgtgccgaac
     1681 gctggaaagc ggaaaatcag gaagggatgg ctgaggtcgc ccggtttatt gaaatgaacg
     1741 gctcttttgc tgacgagaac aggggctggt gaaatgcagt ttaaggttta cacctataaa
     1801 agagagagcc gttatcgtct gtttgtggat gtacagagtg atattattga cacgcccggg
     1861 cgacggatgg tgatccccct ggccagtgca cgtctgctgt cagataaagt ctcccgtgaa
     1921 ctttacccgg tggtgcatat cggggatgaa agctggcgca tgatgaccac cgatatggcc
     1981 agtgtgccgg tttccgttat cggggaagaa gtggctgatc tcagccaccg cgaaaatgac
     2041 atcaaaaacg ccattaacct gatgttctgg ggaatataaa tgtcaggctc ccttatacac
     2101 agccagtcga agacttgttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     2161 atcaacttga aaaagtggca ccgagtcggt gctttttttt cccttgagac caaatcggat
     2221 cccgggcccg tcgactgcag aggcctgcat gcaagcttgg cgtaatcatg gtcatagctg
     2281 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     2341 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     2401 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     2461 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     2521 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     2581 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     2641 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     2701 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     2761 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     2821 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     2881 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     2941 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     3001 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     3061 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     3121 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     3181 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     3241 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     3301 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     3361 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     3421 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     3481 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     3541 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     3601 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     3661 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     3721 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     3781 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     3841 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     3901 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     3961 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     4021 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     4081 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     4141 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     4201 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     4261 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     4321 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     4381 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt
     4441 attatcatga cattaaccta taaaaatagg cgtatcacga ggccctttcg tc
//
