LOCUS       sequence-43053-e        7877 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 7877)
  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 7877)
  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: MIGS1.2 (JW640)
FEATURES             Location/Qualifiers
     source          1..7877
                     /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        2737..3082
                     /label=CaMV 35S promoter
                     /note="strong constitutive promoter from cauliflower mosaic
                     virus"
     primer_bind     3025..3045
                     /label=35S promoter
                     /note="CaMV 35S promoter, forward primer"
     protein_bind    3129..3253
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        join(3278..3283,3284..3301,3302..3308)
                     /label=lac UV5 promoter
                     /note="E. coli lac promoter with an ""up"" mutation"
     CDS             3362..4021
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(3428..3447)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             4363..4668
                     /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     4547..4566
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(4709..4833)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     terminator      4840..5481
                     /label=E9 terminator
                     /note="terminator and polyadenylation signal from the pea 
                     rbcS-E9 gene"
     promoter        complement(5503..5521)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5542..5558)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5542..5558)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5555..5577)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5566..5582
                     /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(5590..5596,5597..5615,5616..5621))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5636..5657
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5774..5791)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5896..5920
                     /label=RB T-DNA repeat
                     /note="right border repeat from nopaline C58 T-DNA"
     rep_origin      complement(6011..6599)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6091..6110)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6770..7558)
                     /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(7849..7877,1..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 ctgcagcccg ggggatccat tcggtcccca gattagcctt ttcaatttca gaaagaatgc
     2281 taacccacag atggttagag aggcttacgc agcaggtctc atcaagacga tctacccgag
     2341 caataatctc caggaaatca aataccttcc caagaaggtt aaagatgcag tcaaaagatt
     2401 caggactaac tgcatcaaga acacagagaa agatatattt ctcaagatca gaagtactat
     2461 tccagtatgg acgattcaag gcttgcttca caaaccaagg caagtaatag agattggagt
     2521 ctctaaaaag gtagttccca ctgaatcaaa ggccatggag tcaaagattc aaatagagga
     2581 cctaacagaa ctcgccgtaa agactggcga acagttcata cagagtctct tacgactcaa
     2641 tgacaagaag aaaatcttcg tcaacatggt ggagcacgac acacttgtct actccaaaaa
     2701 tatcaaagat acagtctcag aagaccaaag ggcaattgag acttttcaac aaagggtaat
     2761 atccggaaac ctcctcggat tccattgccc agctatctgt cactttattg tgaagatagt
     2821 ggaaaaggaa ggtggctcct acaaatgcca tcattgcgat aaaggaaagg ccatcgttga
     2881 agatgcctct gccgacagtg gtcccaaaga tggaccccca cccacgagga gcatcgtgga
     2941 aaaagaagac gttccaacca cgtcttcaaa gcaagtggat tgatgtgata tctccactga
     3001 cgtaagggat gacgcacaat cccactatcc ttcgcaagac ccttcctcta tataaggaag
     3061 ttcatttcat ttggagagaa cacgggggac gagctgtgat ttttctctac aagcgaatct
     3121 agaggatcac aagtttgtac aaaaaagctg aacgagaaac gtaaaatgat ataaatatca
     3181 atatattaaa ttagattttg cataaaaaac agactacata atactgtaaa acacaacata
     3241 tccagtcata ttggcggccc cattaggcac cccaggcttt acactttatg cttccggctc
     3301 gtataatgtg tggattttga gttaggagcc gtcgagattt tcaggagcta aggaagctaa
     3361 aatggagaaa aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga
     3421 acattttgag gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga
     3481 tattacggcc tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat
     3541 tcacattctt gcccgcctga tgaatgctca tccggagttc cgtatggcaa tgaaagacgg
     3601 tgagctggtg atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga
     3661 aacgttttca tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata
     3721 ttcgcaagat gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga
     3781 gaatatgttt ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt
     3841 ggccaatatg gacaacttct tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg
     3901 cgacaaggtg ctgatgccgc tggcgattca ggttcatcat gccgtttgtg atggcttcca
     3961 tgtcggcaga atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta
     4021 aacgcgtgga gccggcttac taaaagccag ataacagtat gcgtatttgc gcgctgattt
     4081 ttgcggtata agaatatata ctgatatgta tacccgaagt atgtcaaaaa gaggtatgct
     4141 atgaagcagc gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat
     4201 atgatgtcaa tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc
     4261 gtgccgaacg ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg
     4321 aaatgaacgg ctcttttgct gacgagaaca ggggctggtg aaatgcagtt taaggtttac
     4381 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac
     4441 acgcccggcc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc
     4501 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc
     4561 gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc
     4621 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggctcc
     4681 cttatacaca gccagtctgc acctcgacca tagtgactgg atatgttgtg ttttacagta
     4741 ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta tatcatttta
     4801 cgtttctcgt tcagctttct tgtacaaagt ggtgatccta gctttcgttc gtatcatcgg
     4861 tttcgacaac gttcgtcaag ttcaatgcat cagtttcatt gcgcacacac cagaatccta
     4921 ctgagtttga gtattatggc attgggaaaa ctgtttttct tgtaccattt gttgtgcttg
     4981 taatttactg tgttttttat tcggttttcg ctatcgaact gtgaaatgga aatggatgga
     5041 gaagagttaa tgaatgatat ggtccttttg ttcattctca aattaatatt atttgttttt
     5101 tctcttattt gttgtgtgtt gaatttgaaa ttataagaga tatgcaaaca ttttgttttg
     5161 agtaaaaatg tgtcaaatcg tggcctctaa tgaccgaagt taatatgagg agtaaaacac
     5221 ttgtagttgt accattatgc ttattcacta ggcaacaaat atattttcag acctagaaaa
     5281 gctgcaaatg ttactgaata caagtatgtc ctcttgtgtt ttagacattt atgaactttc
     5341 ctttatgtaa ttttccagaa tccttgtcag attctaatca ttgctttata attatagtta
     5401 tactcatgga tttgtagttg agtatgaaaa tattttttaa tgcattttat gacttgccaa
     5461 ttgattgaca acatgcatca atcggagctc cagcttttgt tccctttagt gagggttaat
     5521 tccgagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     5581 aattccacac aacatacgag ccggaaghca taaagtgtaa agcctggggt gcctaatgag
     5641 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     5701 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     5761 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5821 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5881 agaacatgaa ggccttgaca ggatatattg gcgggtaaac taagtcgctg tatgtgtttg
     5941 tttgagatct catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     6001 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6061 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6121 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6181 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6241 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6301 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6361 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6421 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6481 agccagttac cttcggaaga agagttggta gctcttgatc cggcaaacaa accaccgctg
     6541 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6601 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6661 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6721 gaagttttaa atcaatctaa agtatatatg tgtaacattg gtctagtgat tatttgccga
     6781 ctaccttggt gatctcgcct ttcacgtagt gaacaaattc ttccaactga tctgcgcgcg
     6841 aggccaagcg atcttcttgt ccaagataag cctgcctagc ttcaagtatg acgggctgat
     6901 actgggccgg caggcgctcc attgcccagt cggcagcgac atccttcggc gcgattttgc
     6961 cggttactgc gctgtaccaa atgcgggaca acgtaagcac tacatttcgc tcatcgccag
     7021 cccagtcggg cggcgagttc catagcgtta aggtttcatt tagcgcctca aatagatcct
     7081 gttcaggaac cggatcaaag agttcctccg ccgctggacc taccaaggca acgctatgtt
     7141 ctcttgcttt tgtcagcaag atagccagat caatgtcgat cgtggctggc tcgaagatac
     7201 ctgcaagaat gtcattgcgc tgccattctc caaattgcag ttcgcgctta gctggataac
     7261 gccacggaat gatgtcgtcg tgcacaacaa tggtgacttc tacagcgcgg agaatctcgc
     7321 tctctccagg ggaagccgaa gtttccaaaa ggtcgttgat caaagctcgc cgcgttgttt
     7381 catcaagcct tacggtcacc gtaaccagca aatcaatatc actgtgtggc ttcaggccgc
     7441 catccactgc ggagccgtac aaatgtacgg ccagcaacgt cggttcgaga tggcgctcga
     7501 tgacgccaac tacctctgat agttgagtcg atacttcggc gatcaccgct tccctcataa
     7561 caccccttgt attactgttt atgtaagcag acagttttat tgttcatgat gatatatttt
     7621 tatcttgtgc aatgtaacat cagagatttt gagacacaac gtggctttgt tgaataaatc
     7681 gaacttttgc tgagttgaag gatcagatca cgcatcttcc cgacaacgca gaccgttccg
     7741 tggcaaagca aaagttcaaa atcaccaact ggtccaccta caacaaagct ctcatcaacc
     7801 gtggctccct cactttctgg ctggatgatg gggcgattca ggcgatcccc atccaacagc
     7861 ccgccgtcga gcgggct
//
