LOCUS       sequence-43069-e        8002 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 8002)
  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 8002)
  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: MIGS5.2 (FF576)
FEATURES             Location/Qualifiers
     source          1..8002
                     /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..1654)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERGRTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTQGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(971..990)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1581..1600
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1688..1867)
                     /label=NOS promoter
                     /note="nopaline synthase promoter"
     primer_bind     2099..2121
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2113..2130
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2114..2130
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2140..2159
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2140..2158
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     2183..2199
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     2184..2200
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        2230..2248
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     2230..2247
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     terminator      complement(2281..2988)
                     /label=OCS terminator
                     /note="octopine synthase terminator"
     primer_bind     complement(3232..3248)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3232..3248)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    3254..3378
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3403..3408,3409..3426,3427..3433)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3487..4146
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3553..3572)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4488..4793
                     /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     4672..4691
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4834..4958)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      4965..5606
                     /label=E9 terminator
                     /note="terminator and polyadenylation signal from the pea 
                     rbcS-E9 gene"
     promoter        complement(5628..5646)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5667..5683)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5667..5683)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5680..5702)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5691..5707
                     /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(5715..5721,5722..5740,5741..5746))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5761..5782
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5899..5916)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6021..6045
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(6136..6724)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6216..6235)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6895..7683)
                     /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(7974..8002,1..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 cagaagaact cgtcaagaag gcgatagaag gcgatgcgct
      901 gcgaatcggg agcggcgata ccgtaaagca cgaggaagcg gtcagcccat tcgccgccaa
      961 gctcttcagc aatatcacgg gtagccaacg ctatgtcctg atagcggtcc gccacaccca
     1021 gccggccaca gtcgatgaat ccagaaaagc ggccattttc caccatgata ttcggcaagc
     1081 aggcatcgcc ctgggtcacg acgagatcct cgccgtcggg catccgcgcc ttgagcctgg
     1141 cgaacagttc ggctggcgcg agcccctgat gctcttcgtc cagatcatcc tgatcgacaa
     1201 gaccggcttc catccgagta cgtcctcgct cgatgcgatg tttcgcttgg tggtcgaatg
     1261 ggcaggtagc cggatcaagc gtatgcagcc gccgcattgc atcagccatg atggatactt
     1321 tctcggcagg agcaaggtga gatgacagga gatcctgccc cggcacttcg cccaatagca
     1381 gccagtccct tcccgcttca gtgacaacgt cgagcacagc tgcgcaagga acgcccgtcg
     1441 tggccagcca cgatagccgc gctgcctcgt cttggagttc attcagggca ccggacaggt
     1501 cggtcttgac aaaaagaacc gggcgcccct gcgctgacag ccggaacacg gcggcatcag
     1561 agcagccgat tgtctgttgt gcccagtcat agccgaatag cctctccacc caagcggccg
     1621 gagaacctgc gtgcaatcca tcttgttcaa tcatgcctcg atcgagttga gagtgaatat
     1681 gagactctaa ttggataccg aggggaattt atggaacgtc agtggagcat ttttgacaag
     1741 aaatatttgc tagctgatag tgaccttagg cgacttttga acgcgcaata atggtttctg
     1801 acgtatgtgc ttagctcatt aaactccaga aacccgcggc tcagtggctc cttcaacgtt
     1861 gcggttctgt cagttccaaa cgtaaaacgg cttgtcccgc gtcatcggcg ggggtcataa
     1921 cgtgactccc ttaattctcc gctcatgatc gataacatta acgcttacaa tttccattcg
     1981 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     2041 cagctggcga aagggggatg tgctgcaagg cgattaagtt gggtaacgcc agggttttcc
     2101 cagtcacgac gttgtaaaac gacggccagt gagcgcgcgt aatacgactc actatagggc
     2161 gaattgggta ccgggccccc cctcgaggtc gacggtatcg ataagcttga tatcgaattc
     2221 ctgcagccca tttaggtgac actatagaat atcactagta agctagcttg catgccggtc
     2281 ctgctgagcc tcgacatgtt gtcgcaaaat tcgccctgga cccgcccaac gatttgtcgt
     2341 cactgtcaag gtttgacctg cacttcattt ggggcccaca tacaccaaaa aaatgctgca
     2401 taattctcgg ggcagcaagt cggttacccg gccgccgtgc tggaccgggt tgaatggtgc
     2461 ccgtaacttt cggtagagcg gacggccaat actcaacttc aaggaatctc acccatgcgc
     2521 gccggcgggg aaccggagtt cccttcagtg agcgttatta gttcgccgct cggtgtgtcg
     2581 tagatactag cccctggggc acttttgaaa tttgaataag atttatgtaa tcagtctttt
     2641 aggtttgacc ggttctgccg ctttttttaa aattggattt gtaataataa aacgcaattg
     2701 tttgttattg tggcgctcta tcatagatgt cgctataaac ctattcagca caatatattg
     2761 ttttcatttt aatattgtac atataagtag tagggtacaa tcagtaaatt gaacggagaa
     2821 tattattcat aaaaatacga tagtaacggg tgatatattc attagaatga accgaaaccg
     2881 gcggtaagga tctgagctac acatgctcag gttttttaca acgtgcacaa cagaattgaa
     2941 agcaaatatc atgcgatcat aggcgtctcg catatctcat taaagcaggg atcccgcatc
     3001 agatctatca aacacataga tttacgaaga tctctaacat taaatcatca gagagataag
     3061 tttagggagc aagctctttc gcttacacag agaatcacag aggaaagaga ctttaagaaa
     3121 actacaactt ttttgaccac tgtgatttct ctctgcaagc gaaagtactt aatcaccaac
     3181 acagtagatt gatatatttg ggtttatttg tgagaagaag aattcttggg ggatccacta
     3241 gttctagagg atcacaagtt tgtacaaaaa agctgaacga gaaacgtaaa atgatataaa
     3301 tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact gtaaaacaca
     3361 acatatccag tcatattggc ggccccatta ggcaccccag gctttacact ttatgcttcc
     3421 ggctcgtata atgtgtggat tttgagttag gagccgtcga gattttcagg agctaaggaa
     3481 gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt
     3541 aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag
     3601 ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc
     3661 tttattcaca ttcttgcccg cctgatgaat gctcatccgg agttccgtat ggcaatgaaa
     3721 gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa
     3781 actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac
     3841 atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt
     3901 attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta
     3961 aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg
     4021 caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc
     4081 ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg gcagggcggg
     4141 gcgtaaacgc gtggagccgg cttactaaaa gccagataac agtatgcgta tttgcgcgct
     4201 gatttttgcg gtataagaat atatactgat atgtataccc gaagtatgtc aaaaagaggt
     4261 atgctatgaa gcagcgtatt acagtgacag ttgacagcga cagctatcag ttgctcaagg
     4321 catatatgat gtcaatatct ccggtctggt aagcacaacc atgcagaatg aagcccgtcg
     4381 tctgcgtgcc gaacgctgga aagcggaaaa tcaggaaggg atggctgagg tcgcccggtt
     4441 tattgaaatg aacggctctt ttgctgacga gaacaggggc tggtgaaatg cagtttaagg
     4501 tttacaccta taaaagagag agccgttatc gtctgtttgt ggatgtacag agtgatatta
     4561 ttgacacgcc cggccgacgg atggtgatcc ccctggccag tgcacgtctg ctgtcagata
     4621 aagtctcccg tgaactttac ccggtggtgc atatcgggga tgaaagctgg cgcatgatga
     4681 ccaccgatat ggccagtgtg ccggtctccg ttatcgggga agaagtggct gatctcagcc
     4741 accgcgaaaa tgacatcaaa aacgccatta acctgatgtt ctggggaata taaatgtcag
     4801 gctcccttat acacagccag tctgcacctc gaccatagtg actggatatg ttgtgtttta
     4861 cagtattatg tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca
     4921 ttttacgttt ctcgttcagc tttcttgtac aaagtggtga tcctagcttt cgttcgtatc
     4981 atcggtttcg acaacgttcg tcaagttcaa tgcatcagtt tcattgcgca cacaccagaa
     5041 tcctactgag tttgagtatt atggcattgg gaaaactgtt tttcttgtac catttgttgt
     5101 gcttgtaatt tactgtgttt tttattcggt tttcgctatc gaactgtgaa atggaaatgg
     5161 atggagaaga gttaatgaat gatatggtcc ttttgttcat tctcaaatta atattatttg
     5221 ttttttctct tatttgttgt gtgttgaatt tgaaattata agagatatgc aaacattttg
     5281 ttttgagtaa aaatgtgtca aatcgtggcc tctaatgacc gaagttaata tgaggagtaa
     5341 aacacttgta gttgtaccat tatgcttatt cactaggcaa caaatatatt ttcagaccta
     5401 gaaaagctgc aaatgttact gaatacaagt atgtcctctt gtgttttaga catttatgaa
     5461 ctttccttta tgtaattttc cagaatcctt gtcagattct aatcattgct ttataattat
     5521 agttatactc atggatttgt agttgagtat gaaaatattt tttaatgcat tttatgactt
     5581 gccaattgat tgacaacatg catcaatcgg agctccagct tttgttccct ttagtgaggg
     5641 ttaattccga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     5701 ctcacaattc cacacaacat acgagccgga aghcataaag tgtaaagcct ggggtgccta
     5761 atgagtgagc taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa
     5821 cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5881 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5941 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     6001 aggaaagaac atgaaggcct tgacaggata tattggcggg taaactaagt cgctgtatgt
     6061 gtttgtttga gatctcatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     6121 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     6181 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     6241 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     6301 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     6361 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     6421 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     6481 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6541 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     6601 gctgaagcca gttaccttcg gaagaagagt tggtagctct tgatccggca aacaaaccac
     6661 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     6721 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     6781 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     6841 aaaatgaagt tttaaatcaa tctaaagtat atatgtgtaa cattggtcta gtgattattt
     6901 gccgactacc ttggtgatct cgcctttcac gtagtgaaca aattcttcca actgatctgc
     6961 gcgcgaggcc aagcgatctt cttgtccaag ataagcctgc ctagcttcaa gtatgacggg
     7021 ctgatactgg gccggcaggc gctccattgc ccagtcggca gcgacatcct tcggcgcgat
     7081 tttgccggtt actgcgctgt accaaatgcg ggacaacgta agcactacat ttcgctcatc
     7141 gccagcccag tcgggcggcg agttccatag cgttaaggtt tcatttagcg cctcaaatag
     7201 atcctgttca ggaaccggat caaagagttc ctccgccgct ggacctacca aggcaacgct
     7261 atgttctctt gcttttgtca gcaagatagc cagatcaatg tcgatcgtgg ctggctcgaa
     7321 gatacctgca agaatgtcat tgcgctgcca ttctccaaat tgcagttcgc gcttagctgg
     7381 ataacgccac ggaatgatgt cgtcgtgcac aacaatggtg acttctacag cgcggagaat
     7441 ctcgctctct ccaggggaag ccgaagtttc caaaaggtcg ttgatcaaag ctcgccgcgt
     7501 tgtttcatca agccttacgg tcaccgtaac cagcaaatca atatcactgt gtggcttcag
     7561 gccgccatcc actgcggagc cgtacaaatg tacggccagc aacgtcggtt cgagatggcg
     7621 ctcgatgacg ccaactacct ctgatagttg agtcgatact tcggcgatca ccgcttccct
     7681 cataacaccc cttgtattac tgtttatgta agcagacagt tttattgttc atgatgatat
     7741 atttttatct tgtgcaatgt aacatcagag attttgagac acaacgtggc tttgttgaat
     7801 aaatcgaact tttgctgagt tgaaggatca gatcacgcat cttcccgaca acgcagaccg
     7861 ttccgtggca aagcaaaagt tcaaaatcac caactggtcc acctacaaca aagctctcat
     7921 caaccgtggc tccctcactt tctggctgga tgatggggcg attcaggcga tccccatcca
     7981 acagcccgcc gtcgagcggg ct
//
