LOCUS       sequence-43064-e        7773 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 7773)
  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 7773)
  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: MIGS5.1 (FF572)
FEATURES             Location/Qualifiers
     source          1..7773
                     /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        2000..2018
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2000..2017
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     terminator      complement(2051..2758)
                     /label=OCS terminator
                     /note="octopine synthase terminator"
     primer_bind     complement(3003..3019)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3003..3019)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    3025..3149
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3174..3179,3180..3197,3198..3204)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3258..3917
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3324..3343)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4259..4564
                     /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     4443..4462
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4605..4729)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      4736..5377
                     /label=E9 terminator
                     /note="terminator and polyadenylation signal from the pea 
                     rbcS-E9 gene"
     promoter        complement(5399..5417)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5438..5454)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5438..5454)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5451..5473)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5462..5478
                     /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(5486..5492,5493..5511,5512..5517))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5532..5553
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5670..5687)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5792..5816
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(5907..6495)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5987..6006)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6666..7454)
                     /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(7745..7773,1..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 ctgcagccca tttaggtgac actatagaat atcactagta agctagcttg
     2041 catgccggtc ctgctgagcc tcgacatgtt gtcgcaaaat tcgccctgga cccgcccaac
     2101 gatttgtcgt cactgtcaag gtttgacctg cacttcattt ggggcccaca tacaccaaaa
     2161 aaatgctgca taattctcgg ggcagcaagt cggttacccg gccgccgtgc tggaccgggt
     2221 tgaatggtgc ccgtaacttt cggtagagcg gacggccaat actcaacttc aaggaatctc
     2281 acccatgcgc gccggcgggg aaccggagtt cccttcagtg agcgttatta gttcgccgct
     2341 cggtgtgtcg tagatactag cccctggggc acttttgaaa tttgaataag atttatgtaa
     2401 tcagtctttt aggtttgacc ggttctgccg ctttttttaa aattggattt gtaataataa
     2461 aacgcaattg tttgttattg tggcgctcta tcatagatgt cgctataaac ctattcagca
     2521 caatatattg ttttcatttt aatattgtac atataagtag tagggtacaa tcagtaaatt
     2581 gaacggagaa tattattcat aaaaatacga tagtaacggg tgatatattc attagaatga
     2641 accgaaaccg gcggtaagga tctgagctac acatgctcag gttttttaca acgtgcacaa
     2701 cagaattgaa agcaaatatc atgcgatcat aggcgtctcg catatctcat taaagcaggg
     2761 atcccgcatc agatctatca aacacataga tttacgaaga tctctaacat taaatcatca
     2821 gagagataag tttagggagc aagctctttc gcttacacag agaatcacag aggaaagaga
     2881 ctttaagaaa actacaactt ttttgaccac tgtgatttct ctctgcaagc gaaagtactt
     2941 aatcaccaac acagtagatt gatatatttg ggtttatttg tgagaagaag aattctttgg
     3001 gggatccact agttctagag gatcacaagt ttgtacaaaa aagctgaacg agaaacgtaa
     3061 aatgatataa atatcaatat attaaattag attttgcata aaaaacagac tacataatac
     3121 tgtaaaacac aacatatcca gtcatattgg cggccccatt aggcacccca ggctttacac
     3181 tttatgcttc cggctcgtat aatgtgtgga ttttgagtta ggagccgtcg agattttcag
     3241 gagctaagga agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc
     3301 aatggcatcg taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc
     3361 agaccgttca gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt
     3421 tttatccggc ctttattcac attcttgccc gcctgatgaa tgctcatccg gagttccgta
     3481 tggcaatgaa agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt
     3541 tccatgagca aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc
     3601 agtttctaca catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc
     3661 ctaaagggtt tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca
     3721 gttttgattt aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca
     3781 aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg
     3841 tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt
     3901 ggcagggcgg ggcgtaaacg cgtggagccg gcttactaaa agccagataa cagtatgcgt
     3961 atttgcgcgc tgatttttgc ggtataagaa tatatactga tatgtatacc cgaagtatgt
     4021 caaaaagagg tatgctatga agcagcgtat tacagtgaca gttgacagcg acagctatca
     4081 gttgctcaag gcatatatga tgtcaatatc tccggtctgg taagcacaac catgcagaat
     4141 gaagcccgtc gtctgcgtgc cgaacgctgg aaagcggaaa atcaggaagg gatggctgag
     4201 gtcgcccggt ttattgaaat gaacggctct tttgctgacg agaacagggg ctggtgaaat
     4261 gcagtttaag gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca
     4321 gagtgatatt attgacacgc ccggccgacg gatggtgatc cccctggcca gtgcacgtct
     4381 gctgtcagat aaagtctccc gtgaacttta cccggtggtg catatcgggg atgaaagctg
     4441 gcgcatgatg accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc
     4501 tgatctcagc caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat
     4561 ataaatgtca ggctccctta tacacagcca gtctgcacct cgaccatagt gactggatat
     4621 gttgtgtttt acagtattat gtagtctgtt ttttatgcaa aatctaattt aatatattga
     4681 tatttatatc attttacgtt tctcgttcag ctttcttgta caaagtggtg atcctagctt
     4741 tcgttcgtat catcggtttc gacaacgttc gtcaagttca atgcatcagt ttcattgcgc
     4801 acacaccaga atcctactga gtttgagtat tatggcattg ggaaaactgt ttttcttgta
     4861 ccatttgttg tgcttgtaat ttactgtgtt ttttattcgg ttttcgctat cgaactgtga
     4921 aatggaaatg gatggagaag agttaatgaa tgatatggtc cttttgttca ttctcaaatt
     4981 aatattattt gttttttctc ttatttgttg tgtgttgaat ttgaaattat aagagatatg
     5041 caaacatttt gttttgagta aaaatgtgtc aaatcgtggc ctctaatgac cgaagttaat
     5101 atgaggagta aaacacttgt agttgtacca ttatgcttat tcactaggca acaaatatat
     5161 tttcagacct agaaaagctg caaatgttac tgaatacaag tatgtcctct tgtgttttag
     5221 acatttatga actttccttt atgtaatttt ccagaatcct tgtcagattc taatcattgc
     5281 tttataatta tagttatact catggatttg tagttgagta tgaaaatatt ttttaatgca
     5341 ttttatgact tgccaattga ttgacaacat gcatcaatcg gagctccagc ttttgttccc
     5401 tttagtgagg gttaattccg agcttggcgt aatcatggtc atagctgttt cctgtgtgaa
     5461 attgttatcc gctcacaatt ccacacaaca tacgagccgg aaghcataaa gtgtaaagcc
     5521 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     5581 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5641 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5701 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5761 ggggataacg caggaaagaa catgaaggcc ttgacaggat atattggcgg gtaaactaag
     5821 tcgctgtatg tgtttgtttg agatctcatg tgagcaaaag gccagcaaaa ggccaggaac
     5881 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     5941 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     6001 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     6061 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     6121 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     6181 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     6241 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     6301 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     6361 atctgcgctc tgctgaagcc agttaccttc ggaagaagag ttggtagctc ttgatccggc
     6421 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     6481 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     6541 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     6601 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgtgta acattggtct
     6661 agtgattatt tgccgactac cttggtgatc tcgcctttca cgtagtgaac aaattcttcc
     6721 aactgatctg cgcgcgaggc caagcgatct tcttgtccaa gataagcctg cctagcttca
     6781 agtatgacgg gctgatactg ggccggcagg cgctccattg cccagtcggc agcgacatcc
     6841 ttcggcgcga ttttgccggt tactgcgctg taccaaatgc gggacaacgt aagcactaca
     6901 tttcgctcat cgccagccca gtcgggcggc gagttccata gcgttaaggt ttcatttagc
     6961 gcctcaaata gatcctgttc aggaaccgga tcaaagagtt cctccgccgc tggacctacc
     7021 aaggcaacgc tatgttctct tgcttttgtc agcaagatag ccagatcaat gtcgatcgtg
     7081 gctggctcga agatacctgc aagaatgtca ttgcgctgcc attctccaaa ttgcagttcg
     7141 cgcttagctg gataacgcca cggaatgatg tcgtcgtgca caacaatggt gacttctaca
     7201 gcgcggagaa tctcgctctc tccaggggaa gccgaagttt ccaaaaggtc gttgatcaaa
     7261 gctcgccgcg ttgtttcatc aagccttacg gtcaccgtaa ccagcaaatc aatatcactg
     7321 tgtggcttca ggccgccatc cactgcggag ccgtacaaat gtacggccag caacgtcggt
     7381 tcgagatggc gctcgatgac gccaactacc tctgatagtt gagtcgatac ttcggcgatc
     7441 accgcttccc tcataacacc ccttgtatta ctgtttatgt aagcagacag ttttattgtt
     7501 catgatgata tatttttatc ttgtgcaatg taacatcaga gattttgaga cacaacgtgg
     7561 ctttgttgaa taaatcgaac ttttgctgag ttgaaggatc agatcacgca tcttcccgac
     7621 aacgcagacc gttccgtggc aaagcaaaag ttcaaaatca ccaactggtc cacctacaac
     7681 aaagctctca tcaaccgtgg ctccctcact ttctggctgg atgatggggc gattcaggcg
     7741 atccccatcc aacagcccgc cgtcgagcgg gct
//
