LOCUS       sequence-115787-        7830 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of amiR-eGFP with a tdTomato gene from the 
            broadly active CAG promoter/enhancer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7830)
  AUTHORS   Miura H, Inoko H, Tanaka M, Nakaoka H, Kimura M, Gurumurthy CB, Sato
            M, Ohtsuka M
  TITLE     Assessment of Artificial MiRNA Architectures for Higher Knockdown 
            Efficiencies without the Undesired Effects in Mice.
  JOURNAL   PLoS One. 2015 Aug 18;10(8):e0135919. doi: 
            10.1371/journal.pone.0135919. eCollection 2015.
  PUBMED    26285215
REFERENCE   2  (bases 1 to 7830)
  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: "PLoS One.";
            date: "2015-08-18"; volume: "10(8):e0135919. doi"; pages: " 
            10.1371/journal.pone.0135919. eCollection 2015"
COMMENT     Sequence Label: pATN (pKD-G12)
FEATURES             Location/Qualifiers
     source          1..7830
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     enhancer        320..623
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        625..900
                     /label=chicken beta-actin promoter
     ncRNA           1277..1303
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           1367..1408
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           1415..1441
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           1505..1546
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     primer_bind     2165..2187
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      2309..2318
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2315..3745
                     /codon_start=1
                     /product="tandem dimeric (pseudo-monomeric) derivative of 
                     DsRed (Shaner et al., 2004)"
                     /label=tdTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLGHGTGSTGSGSSGTASSEDNNMAVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPY
                     EGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVM
                     NFEDGGLVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGV
                     LKGEIHQALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYE
                     RSEGRHHLFLYGMDELYK"
     intron          3779..4351
                     /label=beta-globin intron
                     /note="intron from rabbit beta-globin gene"
     primer_bind     complement(4483..4502)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4548..4603
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4602..4621)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4959..4976)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4959..4975)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4968..4990)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5188..5643
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5275..5294)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5485..5506
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5660..5679)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5779..5801
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5839..5857)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5925..6029
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6030..6098,6099..6890)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6248..6267)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7061..7649
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7550..7569
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7803..7820
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagcttg
      241 catgcctgca ggtcgactct agttattaat agtaatcaat tacggggtca ttagttcata
      301 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      361 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      421 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      481 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      541 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      601 tattagtcat cgctattacc atggtcgagg tgagccccac gttctgcttc actctcccca
      661 tctccccccc ctccccaccc ccaattttgt atttatttat tttttaatta ttttgtgcag
      721 cgatgggggc gggggggggg ggggcgcgcg ccaggcgggg cggggcgggg cgaggggcgg
      781 ggcggggcga ggcggagagg tgcggcggca gccaatcaga gcggcgcgct ccgaaagttt
      841 ccttttatgg cgaggcggcg gcggcggcgg ccctataaaa agcgaagcgc gcggcgggcg
      901 ggagtcgctg cgttgccttc gccccgtgcc ccgctccgcg ccgcctcgcg ccgcccgccc
      961 cggctctgac tgaccgcgtt actcccacag gtgagcgggc gggacggccc ttctcctccg
     1021 ggctgtaatt agcgcttggt ttaatgacgg ctcgtttctt ttctgtggct gcgtgaaagc
     1081 cttaaagggc tccgggaggg ccctttgtgc gggggggagc ggctcggggg gtgcgtgcgt
     1141 gtgtgtgtgc gtggggagcg ccgcgtgcgg cccgcgctgc ccggcggctg tgagcgctgc
     1201 gggcgcggcg cggggctttg tgcgctccgc gtgtgcgcga ggggagcgcg gccgctagtg
     1261 agtcgaccag tggatcctgg aggcttgctg aaggctgtat gctgtgtagt tgtactccag
     1321 cttgtgtttt ggccactgac tgacacaagc tggtacaact acacaggaca caaggcctgt
     1381 tactagcact cacatggaac aaatggccca gatcctggag gcttgctgaa ggctgtatgc
     1441 tgatgaactt cagggtcagc ttggttttgg ccactgactg accaagctga ctgaagttca
     1501 tcaggacaca aggcctgtta ctagcactca catggaacaa atggcccaga tctggccgca
     1561 ctcgagatat ctagacccag cggccggggg cggtgccccg cggtgcgggg gggctgcgag
     1621 gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg
     1681 gcggtcgggc tgtaaccccc ccctgcaccc ccctccccga gttgctgagc acggcccggc
     1741 ttcgggtgcg gggctccgtg cggggcgtgg cgcggggctc gccgtgccgg gcggggggtg
     1801 gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg gccggggagg gctcggggga
     1861 ggggcgcggc ggccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc
     1921 cttttatggt aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctggcggagc
     1981 cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg cgggcgaagc ggtgcggcgc
     2041 cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg ccgcgccgcc gtccccttct
     2101 ccatctccag cctcggggct gccgcagggg gacggctgcc ttcggggggg acggggcagg
     2161 gcggggttcg gcttctggcg tgtgaccggc ggctctagag cctctgctaa ccatgttcat
     2221 gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttattgtgct gtctcatcat
     2281 tttggcaaag aattgggatc caccggtcgc caccatggtg agcaagggcg aggaggtcat
     2341 caaagagttc atgcgcttca aggtgcgcat ggagggctcc atgaacggcc acgagttcga
     2401 gatcgagggc gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt
     2461 gaccaagggc ggccccctgc ccttcgcctg ggacatcctg tccccccagt tcatgtacgg
     2521 ctccaaggcg tacgtgaagc accccgccga catccccgat tacaagaagc tgtccttccc
     2581 cgagggcttc aagtgggagc gcgtgatgaa cttcgaggac ggcggtctgg tgaccgtgac
     2641 ccaggactcc tccctgcagg acggcacgct gatctacaag gtgaagatgc gcggcaccaa
     2701 cttccccccc gacggccccg taatgcagaa gaagaccatg ggctgggagg cctccaccga
     2761 gcgcctgtac ccccgcgacg gcgtgctgaa gggcgagatc caccaggccc tgaagctgaa
     2821 ggacggcggc cactacctgg tggagttcaa gaccatctac atggccaaga agcccgtgca
     2881 actgcccggc tactactacg tggacaccaa gctggacatc acctcccaca acgaggacta
     2941 caccatcgtg gaacagtacg agcgctccga gggccgccac cacctgttcc tggggcatgg
     3001 caccggcagc accggcagcg gcagctccgg caccgcctcc tccgaggaca acaacatggc
     3061 cgtcatcaaa gagttcatgc gcttcaaggt gcgcatggag ggctccatga acggccacga
     3121 gttcgagatc gagggcgagg gcgagggccg cccctacgag ggcacccaga ccgccaagct
     3181 gaaggtgacc aagggcggcc ccctgccctt cgcctgggac atcctgtccc cccagttcat
     3241 gtacggctcc aaggcgtacg tgaagcaccc cgccgacatc cccgattaca agaagctgtc
     3301 cttccccgag ggcttcaagt gggagcgcgt gatgaacttc gaggacggcg gtctggtgac
     3361 cgtgacccag gactcctccc tgcaggacgg cacgctgatc tacaaggtga agatgcgcgg
     3421 caccaacttc ccccccgacg gccccgtaat gcagaagaag accatgggct gggaggcctc
     3481 caccgagcgc ctgtaccccc gcgacggcgt gctgaagggc gagatccacc aggccctgaa
     3541 gctgaaggac ggcggccact acctggtgga gttcaagacc atctacatgg ccaagaagcc
     3601 cgtgcaactg cccggctact actacgtgga caccaagctg gacatcacct cccacaacga
     3661 ggactacacc atcgtggaac agtacgagcg ctccgagggc cgccaccacc tgttcctgta
     3721 cggcatggac gagctgtaca agtaaagtct agattcgaag gatcctgaga acttcagggt
     3781 gagtttgggg acccttgatt gttctttctt tttcgctatt gtaaaattca tgttatatgg
     3841 agggggcaaa gttttcaggg tgttgtttag aatgggaaga tgtcccttgt atcaccatgg
     3901 accctcatga taattttgtt tctttcactt tctactctgt tgacaaccat tgtctcctct
     3961 tattttcttt tcattttctg taactttttc gttaaacttt agcttgcatt tgtaacgaat
     4021 ttttaaattc acttttgttt atttgtcaga ttgtaagtac tttctctaat cacttttttt
     4081 tcaaggcaat cagggtatat tatattgtac ttcagcacag ttttagagaa caattgttat
     4141 aattaaatga taaggtagaa tatttctgca tataaattct ggctggcgtg gaaatattct
     4201 tattggtaga aacaactaca tcctggtcat catcctgcct ttctctttat ggttacaatg
     4261 atatacactg tttgagatga ggataaaata ctctgagtcc aaaccgggcc cctctgctaa
     4321 ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg ttgttgtgct
     4381 gtctcatcat tttggcaaag aattcactcc tcaggtgcag gctgcctatc agaaggtggt
     4441 ggctggtgtg gccaatgccc tggctcacaa ataccactga gatctttttc cctctgccaa
     4501 aaattatggg gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt
     4561 tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg
     4621 gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc
     4681 catatgctgg ctgccatgaa caaaggtggc tataaagagg tcatcagtat atgaaacagc
     4741 cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag atttttttta
     4801 tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta catgttttac
     4861 tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct cttctcttat
     4921 ggagatccct cgaggctagc ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt
     4981 cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc
     5041 gccagctggc gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc
     5101 ctgaatggcg aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     5161 caccgcatac gtcaaagcaa ccatagtacg cgccctgtag cggcgcatta agcgcggcgg
     5221 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
     5281 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
     5341 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
     5401 atttgggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
     5461 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     5521 ctatctcggg ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     5581 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgtttacaa
     5641 ttttatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     5701 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     5761 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     5821 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     5881 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     5941 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     6001 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     6061 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     6121 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     6181 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     6241 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     6301 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     6361 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     6421 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     6481 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     6541 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     6601 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     6661 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     6721 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     6781 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     6841 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     6901 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     6961 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     7021 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     7081 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     7141 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     7201 atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     7261 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     7321 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     7381 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     7441 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     7501 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     7561 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     7621 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     7681 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     7741 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     7801 gcagcgagtc agtgagcgag gaagcggaag
//
