LOCUS       sequence-103237-        6974 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 6974)
  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 6974)
  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: pATG (pKD-G5)
FEATURES             Location/Qualifiers
     source          1..6974
                     /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           3473..3499
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           3563..3604
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           3611..3637
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           3701..3742
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     polyA_signal    3920..4041
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3957..3976)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4011..4030
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4103..4120)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4103..4119)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4112..4134)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4332..4787
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4419..4438)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4629..4650
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4804..4823)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4923..4945
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4983..5001)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        5069..5173
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5174..5242,5243..6034)
                     /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(5392..5411)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6205..6793
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6694..6713
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6947..6964
                     /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 gttgtgctgt 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 taagctaagc
     3421 acttcgtggc cgtcgatcgt ttaaagggag gtagtgagtc gaccagtgga tcctggaggc
     3481 ttgctgaagg ctgtatgctg atgaacttca gggtcagctt ggttttggcc actgactgac
     3541 caagctgact gaagttcatc aggacacaag gcctgttact agcactcaca tggaacaaat
     3601 ggcccagatc ctggaggctt gctgaaggct gtatgctgtg tagttgtact ccagcttgtg
     3661 ttttggccac tgactgacac aagctggtac aactacacag gacacaaggc ctgttactag
     3721 cactcacatg gaacaaatgg cccagatctg gccgcactcg agatatctag acccagcttt
     3781 cttgtacaag taaagcggcc ggccgcgact ctagatcata atcagccata ccacatttgt
     3841 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     3901 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     3961 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     4021 caaactcatc aatgtatctt aaggcgtaaa ttgtaagcgt taatgatcca ctagaggatc
     4081 cccgggtacc gagctcgaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     4141 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     4201 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatggc
     4261 gcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa
     4321 gcaaccatag tacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4381 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4441 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4501 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc
     4561 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     4621 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cgggctattc
     4681 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     4741 acaaaaattt aacgcgaatt ttaacaaaat attaacgttt acaattttat ggtgcactct
     4801 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     4861 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     4921 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     4981 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     5041 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     5101 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     5161 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     5221 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     5281 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     5341 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     5401 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     5461 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     5521 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     5581 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     5641 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     5701 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     5761 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     5821 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     5881 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     5941 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     6001 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     6061 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     6121 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     6181 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     6241 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     6301 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     6361 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     6421 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     6481 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     6541 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     6601 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     6661 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     6721 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     6781 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     6841 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     6901 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     6961 cgaggaagcg gaag
//
