LOCUS       sequence-115959-        4973 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression of amiR-eGFP from the broadly active CAG 
            promoter/enhancer.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4973)
  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 4973)
  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: pARD (pKD-G4)
FEATURES             Location/Qualifiers
     source          1..4973
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        386..661
                     /label=chicken beta-actin promoter
     intron          662..1673
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1597..1619
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1681..1700
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     ncRNA           1749..1775
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           1839..1880
                     /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(1997..2016)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2062..2117
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2116..2135)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2476..2492)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2476..2492)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2489..2511)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2500..2516
                     /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(2524..2530,2531..2548,2549..2554))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2569..2590
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2649..2844
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2695..2830
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2850..2984
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2887..2906)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2941..2960
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3052..3069)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3223..3811)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3303..3322)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3982..4773,4774..4842))
                     /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     4605..4624
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4843..4947)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggact atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atgggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggcgcgc gccaggcggg gcggggcggg gcgaggggcg
      541 gggcggggcg aggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgttgcctt cgccccgtgc cccgctccgc gccgcctcgc gccgcccgcc
      721 ccggctctga ctgaccgcgt tactcccaca ggtgagcggg cgggacggcc cttctcctcc
      781 gggctgtaat tagcgcttgg tttaatgacg gctcgtttct tttctgtggc tgcgtgaaag
      841 ccttaaaggg ctccgggagg gccctttgtg cgggggggag cggctcgggg ggtgcgtgcg
      901 tgtgtgtgtg cgtggggagc gccgcgtgcg gcccgcgctg cccggcggct gtgagcgctg
      961 cgggcgcggc gcggggcttt gtgcgctccg cgtgtgcgcg aggggagcgc ggccgggggc
     1021 ggtgccccgc ggtgcggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg
     1081 tgggggggtg agcagggggt gtgggcgcgg cggtcgggct gtaacccccc cctgcacccc
     1141 cctccccgag ttgctgagca cggcccggct tcgggtgcgg ggctccgtgc ggggcgtggc
     1201 gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg gggtgccggg cggggcgggg
     1261 ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg gccccggagc gccggcggct
     1321 gtcgaggcgc ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg
     1381 gacttccttt gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct
     1441 ctagcgggcg cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc
     1501 ttcgtgcgtc gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg
     1561 gggacggctg ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg
     1621 gcggctctag agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg
     1681 gcaacgtgct ggttattgtg ctgtctcatc attttggcaa agaattctag tgagtcgacc
     1741 agtggatcct ggaggcttgc tgaaggctgt atgctgtgta gttgtactcc agcttgtgtt
     1801 ttggccactg actgacacaa gctggtacaa ctacacagga cacaaggcct gttactagca
     1861 ctcacatgga acaaatggcc cagatctggc cgcactcgag atatctagac ccagaattca
     1921 ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg tgtggccaat gccctggctc
     1981 acaaatacca ctgagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc
     2041 ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga
     2101 attttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga
     2161 atgagtattt ggtttagagt ttggcaacat atgccatatg ctggctgcca tgaacaaagg
     2221 tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc cttattccat
     2281 agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt atttttttct
     2341 ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct cctctcctga
     2401 ctactcccag tcatagctgt ccctcttctc ttatgaagat ccctcgacct gcagcccaag
     2461 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     2521 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     2581 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     2641 gcggatccgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     2701 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     2761 tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg aggaggcttt
     2821 tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat aatggttaca
     2881 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     2941 gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc attaatgaat
     3001 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     3061 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     3121 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     3181 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     3241 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     3301 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     3361 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcaatg
     3421 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     3481 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     3541 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     3601 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     3661 aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     3721 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     3781 gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc
     3841 tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag
     3901 gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata
     3961 tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat
     4021 ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg
     4081 ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc
     4141 tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc
     4201 aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc
     4261 gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc
     4321 gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc
     4381 ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa
     4441 gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat
     4501 gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata
     4561 gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca
     4621 tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag
     4681 gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc
     4741 agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc
     4801 aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata
     4861 ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta
     4921 gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac ctg
//
