LOCUS       sequence-115777-        7828 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 7828)
  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 7828)
  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: pATL (pKD-G10)
FEATURES             Location/Qualifiers
     source          1..7828
                     /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
     intron          901..1909
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1833..1855
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     regulatory      1977..1986
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1983..3413
                     /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"
     ncRNA           3826..3852
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           3916..3957
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           3964..3990
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           4054..4095
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     primer_bind     complement(4481..4500)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4546..4601
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4600..4619)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4957..4974)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4957..4973)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4966..4988)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5186..5641
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5273..5292)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5483..5504
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5658..5677)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5777..5799
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(5837..5855)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5923..6027
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6028..6096,6097..6888)
                     /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(6246..6265)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7059..7647
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7548..7567
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     7801..7818
                     /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 gccgggggcg
     1261 gtgccccgcg gtgcgggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt
     1321 gggggggtga gcagggggtg tgggcgcggc ggtcgggctg taaccccccc ctgcaccccc
     1381 ctccccgagt tgctgagcac ggcccggctt cgggtgcggg gctccgtgcg gggcgtggcg
     1441 cggggctcgc cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc
     1501 cgcctcgggc cggggagggc tcgggggagg ggcgcggcgg ccccggagcg ccggcggctg
     1561 tcgaggcgcg gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg
     1621 acttcctttg tcccaaatct ggcggagccg aaatctggga ggcgccgccg caccccctct
     1681 agcgggcgcg ggcgaagcgg tgcggcgccg gcaggaagga aatgggcggg gagggccttc
     1741 gtgcgtcgcc gcgccgccgt ccccttctcc atctccagcc tcggggctgc cgcaggggga
     1801 cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg tgaccggcgg
     1861 ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc tcctgggcaa
     1921 cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttgggatcca ccggtcgcca
     1981 ccatggtgag caagggcgag gaggtcatca aagagttcat gcgcttcaag gtgcgcatgg
     2041 agggctccat gaacggccac gagttcgaga tcgagggcga gggcgagggc cgcccctacg
     2101 agggcaccca gaccgccaag ctgaaggtga ccaagggcgg ccccctgccc ttcgcctggg
     2161 acatcctgtc cccccagttc atgtacggct ccaaggcgta cgtgaagcac cccgccgaca
     2221 tccccgatta caagaagctg tccttccccg agggcttcaa gtgggagcgc gtgatgaact
     2281 tcgaggacgg cggtctggtg accgtgaccc aggactcctc cctgcaggac ggcacgctga
     2341 tctacaaggt gaagatgcgc ggcaccaact tcccccccga cggccccgta atgcagaaga
     2401 agaccatggg ctgggaggcc tccaccgagc gcctgtaccc ccgcgacggc gtgctgaagg
     2461 gcgagatcca ccaggccctg aagctgaagg acggcggcca ctacctggtg gagttcaaga
     2521 ccatctacat ggccaagaag cccgtgcaac tgcccggcta ctactacgtg gacaccaagc
     2581 tggacatcac ctcccacaac gaggactaca ccatcgtgga acagtacgag cgctccgagg
     2641 gccgccacca cctgttcctg gggcatggca ccggcagcac cggcagcggc agctccggca
     2701 ccgcctcctc cgaggacaac aacatggccg tcatcaaaga gttcatgcgc ttcaaggtgc
     2761 gcatggaggg ctccatgaac ggccacgagt tcgagatcga gggcgagggc gagggccgcc
     2821 cctacgaggg cacccagacc gccaagctga aggtgaccaa gggcggcccc ctgcccttcg
     2881 cctgggacat cctgtccccc cagttcatgt acggctccaa ggcgtacgtg aagcaccccg
     2941 ccgacatccc cgattacaag aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga
     3001 tgaacttcga ggacggcggt ctggtgaccg tgacccagga ctcctccctg caggacggca
     3061 cgctgatcta caaggtgaag atgcgcggca ccaacttccc ccccgacggc cccgtaatgc
     3121 agaagaagac catgggctgg gaggcctcca ccgagcgcct gtacccccgc gacggcgtgc
     3181 tgaagggcga gatccaccag gccctgaagc tgaaggacgg cggccactac ctggtggagt
     3241 tcaagaccat ctacatggcc aagaagcccg tgcaactgcc cggctactac tacgtggaca
     3301 ccaagctgga catcacctcc cacaacgagg actacaccat cgtggaacag tacgagcgct
     3361 ccgagggccg ccaccacctg ttcctgtacg gcatggacga gctgtacaag taaagtctag
     3421 attcgaagga tcctgagaac ttcagggtga gtttggggac ccttgattgt tctttctttt
     3481 tcgctattgt aaaattcatg ttatatggag ggggcaaagt tttcagggtg ttgtttagaa
     3541 tgggaagatg tcccttgtat caccatggac cctcatgata attttgtttc tttcactttc
     3601 tactctgttg acaaccattg tctcctctta ttttcttttc attttctgta actttttcgt
     3661 taaactttag cttgcatttg taacgaattt ttaaattcac ttttgtttat ttgtcagatt
     3721 gtaagtactt tctctaatca cttttttttc aaggcaatca gggtatatta tattgtactt
     3781 cagcacagtt ttagagaaca attctagtga gtcgaccagt ggatcctgga ggcttgctga
     3841 aggctgtatg ctgatgaact tcagggtcag cttggttttg gccactgact gaccaagctg
     3901 actgaagttc atcaggacac aaggcctgtt actagcactc acatggaaca aatggcccag
     3961 atcctggagg cttgctgaag gctgtatgct gtgtagttgt actccagctt gtgttttggc
     4021 cactgactga cacaagctgg tacaactaca caggacacaa ggcctgttac tagcactcac
     4081 atggaacaaa tggcccagat ctggccgcac tcgagatatc tagacccaga attgttataa
     4141 ttaaatgata aggtagaata tttctgcata taaattctgg ctggcgtgga aatattctta
     4201 ttggtagaaa caactacacc ctggtcatca tcctgccttt ctctttatgg ttacaatgat
     4261 atacactgtt tgagatgagg ataaaatact ctgagtccaa accgggcccc tctgctaacc
     4321 atgttcatgc cttcttctct ttcctacagc tcctgggcaa cgtgctggtt gttgtgctgt
     4381 ctcatcattt tggcaaagaa ttcactcctc aggtgcaggc tgcctatcag aaggtggtgg
     4441 ctggtgtggc caatgccctg gctcacaaat accactgaga tctttttccc tctgccaaaa
     4501 attatgggga catcatgaag ccccttgagc atctgacttc tggctaataa aggaaattta
     4561 ttttcattgc aatagtgtgt tggaattttt tgtgtctctc actcggaagg acatatggga
     4621 gggcaaatca tttaaaacat cagaatgagt atttggttta gagtttggca acatatgcca
     4681 tatgctggct gccatgaaca aaggtggcta taaagaggtc atcagtatat gaaacagccc
     4741 cctgctgtcc attccttatt ccatagaaaa gccttgactt gaggttagat tttttttata
     4801 ttttgttttg tgttattttt ttctttaaca tccctaaaat tttccttaca tgttttacta
     4861 gccagatttt tcctcctctc ctgactactc ccagtcatag ctgtccctct tctcttatgg
     4921 agatccctcg aggctagcgg taccgagctc gaattcactg gccgtcgttt tacaacgtcg
     4981 tgactgggaa aaccctggcg ttacccaact taatcgcctt gcagcacatc cccctttcgc
     5041 cagctggcgt aatagcgaag aggcccgcac cgatcgccct tcccaacagt tgcgcagcct
     5101 gaatggcgaa tggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     5161 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     5221 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     5281 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     5341 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     5401 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     5461 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct
     5521 atctcgggct attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa
     5581 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     5641 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     5701 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     5761 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     5821 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     5881 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     5941 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     6001 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     6061 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     6121 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     6181 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     6241 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     6301 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     6361 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     6421 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     6481 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     6541 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     6601 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     6661 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     6721 aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt
     6781 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     6841 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     6901 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     6961 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     7021 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     7081 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     7141 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     7201 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     7261 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     7321 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     7381 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     7441 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     7501 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     7561 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     7621 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     7681 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     7741 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     7801 agcgagtcag tgagcgagga agcggaag
//
