LOCUS       sequence-43060-e        8410 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 8410)
  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 8410)
  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: MIGS4.1 (FF571)
FEATURES             Location/Qualifiers
     source          1..8410
                     /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"
     promoter        1943..1961
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     1943..1960
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     terminator      complement(1994..2701)
                     /label=OCS terminator
                     /note="octopine synthase terminator"
     primer_bind     complement(3640..3656)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3640..3656)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    3662..3786
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3811..3816,3817..3834,3835..3841)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3895..4554
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3961..3980)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4896..5201
                     /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     5080..5099
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(5242..5366)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      5373..6014
                     /label=E9 terminator
                     /note="terminator and polyadenylation signal from the pea 
                     rbcS-E9 gene"
     promoter        complement(6036..6054)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6075..6091)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6075..6091)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6088..6110)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6099..6115
                     /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(6123..6129,6130..6148,6149..6154))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6169..6190
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6307..6324)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6429..6453
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(6544..7132)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6624..6643)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7303..8091)
                     /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(8382..8410,1..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 ccatttaggt gacactatag aatatcacta gtaagctagc
     1981 ttgcatgccg gtcctgctga gcctcgacat gttgtcgcaa aattcgccct ggacccgccc
     2041 aacgatttgt cgtcactgtc aaggtttgac ctgcacttca tttggggccc acatacacca
     2101 aaaaaatgct gcataattct cggggcagca agtcggttac ccggccgccg tgctggaccg
     2161 ggttgaatgg tgcccgtaac tttcggtaga gcggacggcc aatactcaac ttcaaggaat
     2221 ctcacccatg cgcgccggcg gggaaccgga gttcccttca gtgagcgtta ttagttcgcc
     2281 gctcggtgtg tcgtagatac tagcccctgg ggcacttttg aaatttgaat aagatttatg
     2341 taatcagtct tttaggtttg accggttctg ccgctttttt taaaattgga tttgtaataa
     2401 taaaacgcaa ttgtttgtta ttgtggcgct ctatcataga tgtcgctata aacctattca
     2461 gcacaatata ttgttttcat tttaatattg tacatataag tagtagggta caatcagtaa
     2521 attgaacgga gaatattatt cataaaaata cgatagtaac gggtgatata ttcattagaa
     2581 tgaaccgaaa ccggcggtaa ggatctgagc tacacatgct caggtttttt acaacgtgca
     2641 caacagaatt gaaagcaaat atcatgcgat cataggcgtc tcgcatatct cattaaagca
     2701 gggatcccgc atcagatcta tcaaacacat agatttacga agatctctaa cattaaatca
     2761 tcagagagat aagtttaggg agcaagctct ttcgcttaca cagagaatca cagaggaaag
     2821 agactttaag aaaactacaa cttttttgac cactgtgatt tctctctgca agcgaaagta
     2881 cttaatcacc aacacagtag attgatatat ttgggtttat ttgtgagaag aagaattcac
     2941 tagtggatcc cgcactcgag tgttaatcag aaaaactcag attaatctac aaattcgatc
     3001 gcacaaacta gaaactaaca ccagatctag atagaaatca caaatcgaag agtaattatt
     3061 cgacaaaact caaattattt gaacaaatcg gatgatatct atgaaaccct aatcgagaat
     3121 taagatgata tctaacgatc aaacccagaa aatcgtcttc gatctaagat taacagaatc
     3181 taaaccaaag aacatatacg aaattgggat cgaacgaaaa caaaatcgaa gattttgaga
     3241 gaataaggaa cacagaaatt taccttgatc acggtagaga gaattgagag aaagttttta
     3301 agattttgag aaattgaaat ctgaattgtg aagaagaaga gctctttggg tattgtttta
     3361 tagaagaaga agaagaaaag acgaggacga ctaggtcacg agaaagctaa ggcggtgaag
     3421 caatagctaa taataaaatg acacgtgtat tgagcgttgt ttacacgcaa agttgttttt
     3481 ggctaattgc cttattttta ggttgaggaa aagtatttgt gctttgagtt gataaacacg
     3541 actcgtgtgt gccggctgca accactttga cgccgtttat tactgactcg tcggtcgacg
     3601 gtatcgataa gcttgatatc gaattcctgc agcccggggg atccactagt tctagaggat
     3661 cacaagtttg tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt
     3721 aaattagatt ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc
     3781 atattggcgg ccccattagg caccccaggc tttacacttt atgcttccgg ctcgtataat
     3841 gtgtggattt tgagttagga gccgtcgaga ttttcaggag ctaaggaagc taaaatggag
     3901 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
     3961 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
     4021 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
     4081 cttgcccgcc tgatgaatgc tcatccggag ttccgtatgg caatgaaaga cggtgagctg
     4141 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
     4201 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
     4261 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
     4321 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
     4381 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
     4441 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc
     4501 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaaacgcgt
     4561 ggagccggct tactaaaagc cagataacag tatgcgtatt tgcgcgctga tttttgcggt
     4621 ataagaatat atactgatat gtatacccga agtatgtcaa aaagaggtat gctatgaagc
     4681 agcgtattac agtgacagtt gacagcgaca gctatcagtt gctcaaggca tatatgatgt
     4741 caatatctcc ggtctggtaa gcacaaccat gcagaatgaa gcccgtcgtc tgcgtgccga
     4801 acgctggaaa gcggaaaatc aggaagggat ggctgaggtc gcccggttta ttgaaatgaa
     4861 cggctctttt gctgacgaga acaggggctg gtgaaatgca gtttaaggtt tacacctata
     4921 aaagagagag ccgttatcgt ctgtttgtgg atgtacagag tgatattatt gacacgcccg
     4981 gccgacggat ggtgatcccc ctggccagtg cacgtctgct gtcagataaa gtctcccgtg
     5041 aactttaccc ggtggtgcat atcggggatg aaagctggcg catgatgacc accgatatgg
     5101 ccagtgtgcc ggtctccgtt atcggggaag aagtggctga tctcagccac cgcgaaaatg
     5161 acatcaaaaa cgccattaac ctgatgttct ggggaatata aatgtcaggc tcccttatac
     5221 acagccagtc tgcacctcga ccatagtgac tggatatgtt gtgttttaca gtattatgta
     5281 gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct
     5341 cgttcagctt tcttgtacaa agtggtgatc ctagctttcg ttcgtatcat cggtttcgac
     5401 aacgttcgtc aagttcaatg catcagtttc attgcgcaca caccagaatc ctactgagtt
     5461 tgagtattat ggcattggga aaactgtttt tcttgtacca tttgttgtgc ttgtaattta
     5521 ctgtgttttt tattcggttt tcgctatcga actgtgaaat ggaaatggat ggagaagagt
     5581 taatgaatga tatggtcctt ttgttcattc tcaaattaat attatttgtt ttttctctta
     5641 tttgttgtgt gttgaatttg aaattataag agatatgcaa acattttgtt ttgagtaaaa
     5701 atgtgtcaaa tcgtggcctc taatgaccga agttaatatg aggagtaaaa cacttgtagt
     5761 tgtaccatta tgcttattca ctaggcaaca aatatatttt cagacctaga aaagctgcaa
     5821 atgttactga atacaagtat gtcctcttgt gttttagaca tttatgaact ttcctttatg
     5881 taattttcca gaatccttgt cagattctaa tcattgcttt ataattatag ttatactcat
     5941 ggatttgtag ttgagtatga aaatattttt taatgcattt tatgacttgc caattgattg
     6001 acaacatgca tcaatcggag ctccagcttt tgttcccttt agtgagggtt aattccgagc
     6061 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     6121 cacaacatac gagccggaag hcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     6181 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     6241 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     6301 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     6361 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     6421 gaaggccttg acaggatata ttggcgggta aactaagtcg ctgtatgtgt ttgtttgaga
     6481 tctcatgtga gcaaaaggcc agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc
     6541 gtttttccat aggctccgcc cccctgacga gcatcacaaa aatcgacgct caagtcagag
     6601 gtggcgaaac ccgacaggac tataaagata ccaggcgttt ccccctggaa gctccctcgt
     6661 gcgctctcct gttccgaccc tgccgcttac cggatacctg tccgcctttc tcccttcggg
     6721 aagcgtggcg ctttctcata gctcacgctg taggtatctc agttcggtgt aggtcgttcg
     6781 ctccaagctg ggctgtgtgc acgaaccccc cgttcagccc gaccgctgcg ccttatccgg
     6841 taactatcgt cttgagtcca acccggtaag acacgactta tcgccactgg cagcagccac
     6901 tggtaacagg attagcagag cgaggtatgt aggcggtgct acagagttct tgaagtggtg
     6961 gcctaactac ggctacacta gaagaacagt atttggtatc tgcgctctgc tgaagccagt
     7021 taccttcgga agaagagttg gtagctcttg atccggcaaa caaaccaccg ctggtagcgg
     7081 tggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc aagaagatcc
     7141 tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt aagggatttt
     7201 ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa aatgaagttt
     7261 taaatcaatc taaagtatat atgtgtaaca ttggtctagt gattatttgc cgactacctt
     7321 ggtgatctcg cctttcacgt agtgaacaaa ttcttccaac tgatctgcgc gcgaggccaa
     7381 gcgatcttct tgtccaagat aagcctgcct agcttcaagt atgacgggct gatactgggc
     7441 cggcaggcgc tccattgccc agtcggcagc gacatccttc ggcgcgattt tgccggttac
     7501 tgcgctgtac caaatgcggg acaacgtaag cactacattt cgctcatcgc cagcccagtc
     7561 gggcggcgag ttccatagcg ttaaggtttc atttagcgcc tcaaatagat cctgttcagg
     7621 aaccggatca aagagttcct ccgccgctgg acctaccaag gcaacgctat gttctcttgc
     7681 ttttgtcagc aagatagcca gatcaatgtc gatcgtggct ggctcgaaga tacctgcaag
     7741 aatgtcattg cgctgccatt ctccaaattg cagttcgcgc ttagctggat aacgccacgg
     7801 aatgatgtcg tcgtgcacaa caatggtgac ttctacagcg cggagaatct cgctctctcc
     7861 aggggaagcc gaagtttcca aaaggtcgtt gatcaaagct cgccgcgttg tttcatcaag
     7921 ccttacggtc accgtaacca gcaaatcaat atcactgtgt ggcttcaggc cgccatccac
     7981 tgcggagccg tacaaatgta cggccagcaa cgtcggttcg agatggcgct cgatgacgcc
     8041 aactacctct gatagttgag tcgatacttc ggcgatcacc gcttccctca taacacccct
     8101 tgtattactg tttatgtaag cagacagttt tattgttcat gatgatatat ttttatcttg
     8161 tgcaatgtaa catcagagat tttgagacac aacgtggctt tgttgaataa atcgaacttt
     8221 tgctgagttg aaggatcaga tcacgcatct tcccgacaac gcagaccgtt ccgtggcaaa
     8281 gcaaaagttc aaaatcacca actggtccac ctacaacaaa gctctcatca accgtggctc
     8341 cctcactttc tggctggatg atggggcgat tcaggcgatc cccatccaac agcccgccgt
     8401 cgagcgggct
//
