LOCUS       sequence-43052-e        7647 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7647)
  AUTHORS   de Felippes FF, Wang JW, Weigel D
  TITLE     MIGS: MiRNA Induced Gene Silencing.
  JOURNAL   Plant J. 2011 Dec 28. doi: 10.1111/j.1365-313X.2011.04896.x.
  PUBMED    22211571
REFERENCE   2  (bases 1 to 7647)
  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. 
            2011 Dec 28. doi: 10.1111/j.1365-313X.2011.04896.x."
COMMENT     Sequence Label: MIGS1.1 (JW493)
FEATURES             Location/Qualifiers
     source          1..7647
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    542..564
                     /label=LB T-DNA repeat
                     /note="left border repeat from nopaline C58 T-DNA 
                     (truncated)"
     terminator      complement(588..840)
                     /label=NOS terminator
                     /note="nopaline synthase terminator and poly(A) signal"
     CDS             complement(860..1411)
                     /codon_start=1
                     /gene="Streptomyces hygroscopicus bar"
                     /product="phosphinothricin acetyltransferase"
                     /label=BlpR
                     /note="confers resistance to bialophos or phosphinothricin"
                     /translation="MSPERRPADIRRATEADMPAVCTIVNHYIETSTVNFRTEPQEPQE
                     WTDDLVRLRERYPWLVAEVDGEVAGIAYAGPWKARNAYDWTAESTVYVSPRHQRTGLGS
                     TLYTHLLKSLEAQGFKSVVAVIGLPNDPSVRMHEALGYAPRGMLRAAGFKHGNWHDVGF
                     WQLDFSLPVPPRPVLPVTEI"
     promoter        complement(1453..1632)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     1869..1891
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1883..1900
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1884..1900
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     1910..1929
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1910..1928
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     1953..1969
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     1954..1970
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2507..2852
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     2795..2815
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     protein_bind    2899..3023
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3048..3053,3054..3071,3072..3078)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3132..3791
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3198..3217)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4133..4438
                     /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     4317..4336
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4479..4603)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      4610..5251
                     /label=E9 terminator
                     /note="terminator and polyadenylation signal from the pea 
                     rbcS-E9 gene"
     promoter        complement(5273..5291)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5312..5328)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5312..5328)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5325..5347)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5336..5352
                     /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(5360..5366,5367..5385,5386..5391))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5406..5427
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5544..5561)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5666..5690
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(5781..6369)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5861..5880)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6540..7328)
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      join(7619..7647,1..407)
                     /label=pSa ori
                     /note="origin of replication from bacterial plasmid pSa"
ORIGIN
        1 tttttatccc cggaagcctg tggatagagg gtagttatcc acgtgaaacc gctaatgccc
       61 cgcaaagcct tgattcacgg ggctttccgg cccgctccaa aaactatcca cgtgaaatcg
      121 ctaatcaggg tacgtgaaat cgctaatcgg agtacgtgaa atcgctaata aggtcacgtg
      181 aaatcgctaa tcaaaaaggc acgtgagaac gctaatagcc ctttcagatc aacagcttgc
      241 aaacacccct cgctccggca agtagttaca gcaagtagta tgttcaatta gcttttcaat
      301 tatgaatata tatatcaatt attggtcgcc cttggcttgt ggacaatgcg ctacgcgcac
      361 cggctccgcc cgtggacaac cgcaagcggt tgcccaccgt cgagcgccag cgcctttgcc
      421 cacaacccgg cggccggccg caacagatcg ttttataaat tttttttttt gaaaaagaaa
      481 aagcccgaaa ggcggcaacc tctcgggctt ctggatttcc gatccccgga attagagatc
      541 ttggcaggat atattgtggt gtaacgttat cagcttgcat gccggtcgat ctagtaacat
      601 agatgacacc gcgcgcgata atttatccta gtttgcgcgc tatattttgt tttctatcgc
      661 gtattaaatg tataattgcg ggactctaat cataaaaacc catctcataa ataacgtcat
      721 gcattacatg ttaattatta catgcttaac gtaattcaac agaaattata tgataatcat
      781 cgcaagaccg gcaacaggat tcaatcttaa gaaactttat tgccaaatgt ttgaacgatc
      841 tgcttgactc taggggtcat cagatttcgg tgacgggcag gaccggacgg ggcggcaccg
      901 gcaggctgaa gtccagctgc cagaaaccca cgtcatgcca gttcccgtgc ttgaagccgg
      961 ccgcccgcag catgccgcgg ggggcatatc cgagcgcctc gtgcatgcgc acgctcgggt
     1021 cgttgggcag cccgatgaca gcgaccacgc tcttgaagcc ctgtgcctcc agggacttca
     1081 gcaggtgggt gtagagcgtg gagcccagtc ccgtccgctg gtggcggggg gagacgtaca
     1141 cggtggactc ggccgtccag tcgtaggcgt tgcgtgcctt ccagggaccc gcgtaggcga
     1201 tgccggcgac ctcgccgtcc acctcggcga cgagccaggg atagcgctcc cgcagacgga
     1261 cgaggtcgtc cgtccactcc tgcggttcct gcggctcggt acggaagttg accgtgcttg
     1321 tctcgatgta gtggttgacg atggtgcaga ccgccggcat gtccgcctcg gtggcacggc
     1381 ggatgtcggc cgggcgtcgt tctgggctca tggtagatcc cctcgatcga gttgagagtg
     1441 aatatgagac tctaattgga taccgagggg aatttatgga acgtcagtgg agcatttttg
     1501 acaagaaata tttgctagct gatagtgacc ttaggcgact tttgaacgcg caataatggt
     1561 ttctgacgta tgtgcttagc tcattaaact ccagaaaccc gcggctcagt ggctccttca
     1621 acgttgcggt tctgtcagtt ccaaacgtaa aacggcttgt cccgcgtcat cggcgggggt
     1681 cataacgtga ctcccttaat tctccgctca tgtatcgata acattaacgt ttacaatttc
     1741 gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc
     1801 gctattacgc cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc
     1861 agggttttcc cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc
     1921 actatagggc gaattgggta ccgggccccc cctcgaggtc gacggtatcg ataagcttga
     1981 tatcgaattc ctgcagcccg ggggatccat tcggtcccca gattagcctt ttcaatttca
     2041 gaaagaatgc taacccacag atggttagag aggcttacgc agcaggtctc atcaagacga
     2101 tctacccgag caataatctc caggaaatca aataccttcc caagaaggtt aaagatgcag
     2161 tcaaaagatt caggactaac tgcatcaaga acacagagaa agatatattt ctcaagatca
     2221 gaagtactat tccagtatgg acgattcaag gcttgcttca caaaccaagg caagtaatag
     2281 agattggagt ctctaaaaag gtagttccca ctgaatcaaa ggccatggag tcaaagattc
     2341 aaatagagga cctaacagaa ctcgccgtaa agactggcga acagttcata cagagtctct
     2401 tacgactcaa tgacaagaag aaaatcttcg tcaacatggt ggagcacgac acacttgtct
     2461 actccaaaaa tatcaaagat acagtctcag aagaccaaag ggcaattgag acttttcaac
     2521 aaagggtaat atccggaaac ctcctcggat tccattgccc agctatctgt cactttattg
     2581 tgaagatagt ggaaaaggaa ggtggctcct acaaatgcca tcattgcgat aaaggaaagg
     2641 ccatcgttga agatgcctct gccgacagtg gtcccaaaga tggaccccca cccacgagga
     2701 gcatcgtgga aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgata
     2761 tctccactga cgtaagggat gacgcacaat cccactatcc ttcgcaagac ccttcctcta
     2821 tataaggaag ttcatttcat ttggagagaa cacgggggac gagctgtgat ttttctctac
     2881 aagcgaatct agaggatcac aagtttgtac aaaaaagctg aacgagaaac gtaaaatgat
     2941 ataaatatca atatattaaa ttagattttg cataaaaaac agactacata atactgtaaa
     3001 acacaacata tccagtcata ttggcggccc cattaggcac cccaggcttt acactttatg
     3061 cttccggctc gtataatgtg tggattttga gttaggagcc gtcgagattt tcaggagcta
     3121 aggaagctaa aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc
     3181 atcgtaaaga acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg
     3241 ttcagctgga tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc
     3301 cggcctttat tcacattctt gcccgcctga tgaatgctca tccggagttc cgtatggcaa
     3361 tgaaagacgg tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg
     3421 agcaaactga aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc
     3481 tacacatata ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag
     3541 ggtttattga gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg
     3601 atttaaacgt ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt
     3661 atacgcaagg cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg
     3721 atggcttcca tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg
     3781 gcggggcgta aacgcgtgga gccggcttac taaaagccag ataacagtat gcgtatttgc
     3841 gcgctgattt ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaaa
     3901 gaggtatgct atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct
     3961 caaggcatat atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc
     4021 cgtcgtctgc gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc
     4081 cggtttattg aaatgaacgg ctcttttgct gacgagaaca ggggctggtg aaatgcagtt
     4141 taaggtttac acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga
     4201 tattattgac acgcccggcc gacggatggt gatccccctg gccagtgcac gtctgctgtc
     4261 agataaagtc tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat
     4321 gatgaccacc gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct
     4381 cagccaccgc gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat
     4441 gtcaggctcc cttatacaca gccagtctgc acctcgacca tagtgactgg atatgttgtg
     4501 ttttacagta ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta
     4561 tatcatttta cgtttctcgt tcagctttct tgtacaaagt ggtgatccta gctttcgttc
     4621 gtatcatcgg tttcgacaac gttcgtcaag ttcaatgcat cagtttcatt gcgcacacac
     4681 cagaatccta ctgagtttga gtattatggc attgggaaaa ctgtttttct tgtaccattt
     4741 gttgtgcttg taatttactg tgttttttat tcggttttcg ctatcgaact gtgaaatgga
     4801 aatggatgga gaagagttaa tgaatgatat ggtccttttg ttcattctca aattaatatt
     4861 atttgttttt tctcttattt gttgtgtgtt gaatttgaaa ttataagaga tatgcaaaca
     4921 ttttgttttg agtaaaaatg tgtcaaatcg tggcctctaa tgaccgaagt taatatgagg
     4981 agtaaaacac ttgtagttgt accattatgc ttattcacta ggcaacaaat atattttcag
     5041 acctagaaaa gctgcaaatg ttactgaata caagtatgtc ctcttgtgtt ttagacattt
     5101 atgaactttc ctttatgtaa ttttccagaa tccttgtcag attctaatca ttgctttata
     5161 attatagtta tactcatgga tttgtagttg agtatgaaaa tattttttaa tgcattttat
     5221 gacttgccaa ttgattgaca acatgcatca atcggagctc cagcttttgt tccctttagt
     5281 gagggttaat tccgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt
     5341 atccgctcac aattccacac aacatacgag ccggaaghca taaagtgtaa agcctggggt
     5401 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5461 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     5521 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     5581 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     5641 aacgcaggaa agaacatgaa ggccttgaca ggatatattg gcgggtaaac taagtcgctg
     5701 tatgtgtttg tttgagatct catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5761 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5821 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5881 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5941 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6001 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6061 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6121 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6181 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6241 gctctgctga agccagttac cttcggaaga agagttggta gctcttgatc cggcaaacaa
     6301 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6361 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6421 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6481 aattaaaaat gaagttttaa atcaatctaa agtatatatg tgtaacattg gtctagtgat
     6541 tatttgccga ctaccttggt gatctcgcct ttcacgtagt gaacaaattc ttccaactga
     6601 tctgcgcgcg aggccaagcg atcttcttgt ccaagataag cctgcctagc ttcaagtatg
     6661 acgggctgat actgggccgg caggcgctcc attgcccagt cggcagcgac atccttcggc
     6721 gcgattttgc cggttactgc gctgtaccaa atgcgggaca acgtaagcac tacatttcgc
     6781 tcatcgccag cccagtcggg cggcgagttc catagcgtta aggtttcatt tagcgcctca
     6841 aatagatcct gttcaggaac cggatcaaag agttcctccg ccgctggacc taccaaggca
     6901 acgctatgtt ctcttgcttt tgtcagcaag atagccagat caatgtcgat cgtggctggc
     6961 tcgaagatac ctgcaagaat gtcattgcgc tgccattctc caaattgcag ttcgcgctta
     7021 gctggataac gccacggaat gatgtcgtcg tgcacaacaa tggtgacttc tacagcgcgg
     7081 agaatctcgc tctctccagg ggaagccgaa gtttccaaaa ggtcgttgat caaagctcgc
     7141 cgcgttgttt catcaagcct tacggtcacc gtaaccagca aatcaatatc actgtgtggc
     7201 ttcaggccgc catccactgc ggagccgtac aaatgtacgg ccagcaacgt cggttcgaga
     7261 tggcgctcga tgacgccaac tacctctgat agttgagtcg atacttcggc gatcaccgct
     7321 tccctcataa caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat
     7381 gatatatttt tatcttgtgc aatgtaacat cagagatttt gagacacaac gtggctttgt
     7441 tgaataaatc gaacttttgc tgagttgaag gatcagatca cgcatcttcc cgacaacgca
     7501 gaccgttccg tggcaaagca aaagttcaaa atcaccaact ggtccaccta caacaaagct
     7561 ctcatcaacc gtggctccct cactttctgg ctggatgatg gggcgattca ggcgatcccc
     7621 atccaacagc ccgccgtcga gcgggct
//
