LOCUS       sequence-115768-        5112 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 5112)
  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 5112)
  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: pATI (pKD-G7)
FEATURES             Location/Qualifiers
     source          1..5112
                     /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..1672
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1596..1618
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1680..1699
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     ncRNA           1748..1774
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           1838..1879
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           1886..1912
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           1976..2017
                     /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(2134..2153)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2199..2254
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2253..2272)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(2615..2631)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2615..2631)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2628..2650)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2639..2655
                     /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(2663..2669,2670..2687,2688..2693))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2708..2729
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        2788..2983
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      2834..2969
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    2989..3123
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3026..3045)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3080..3099
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3191..3208)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3362..3950)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3442..3461)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4121..4912,4913..4981))
                     /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     4744..4763
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4982..5086)
                     /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 tggcggagcc gaaatctggg aggcgccgcc gcaccccctc
     1441 tagcgggcgc ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct
     1501 tcgtgcgtcg ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg
     1561 ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg
     1621 cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg
     1681 caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattctagt gagtcgacca
     1741 gtggatcctg gaggcttgct gaaggctgta tgctgatgaa cttcagggtc agcttggttt
     1801 tggccactga ctgaccaagc tgactgaagt tcatcaggac acaaggcctg ttactagcac
     1861 tcacatggaa caaatggccc agatcctgga ggcttgctga aggctgtatg ctgtgtagtt
     1921 gtactccagc ttgtgttttg gccactgact gacacaagct ggtacaacta cacaggacac
     1981 aaggcctgtt actagcactc acatggaaca aatggcccag atctggccgc actcgagata
     2041 tctagaccca gaattcactc ctcaggtgca ggctgcctat cagaaggtgg tggctggtgt
     2101 ggccaatgcc ctggctcaca aataccactg agatcttttt ccctctgcca aaaattatgg
     2161 ggacatcatg aagccccttg agcatctgac ttctggctaa taaaggaaat ttattttcat
     2221 tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga aggacatatg ggagggcaaa
     2281 tcatttaaaa catcagaatg agtatttggt ttagagtttg gcaacatatg cccatatgct
     2341 ggctgccatg aacaaaggtt ggctataaag aggtcatcag tatatgaaac agccccctgc
     2401 tgtccattcc ttattccata gaaaagcctt gacttgaggt tagatttttt ttatattttg
     2461 ttttgtgtta tttttttctt taacatccct aaaattttcc ttacatgttt tactagccag
     2521 atttttcctc ctctcctgac tactcccagt catagctgtc cctcttctct tatgaagatc
     2581 cctcgacctg cagcccaagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     2641 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     2701 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     2761 cgggaaacct gtcgtgccag cggatccgca tctcaattag tcagcaacca tagtcccgcc
     2821 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     2881 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     2941 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaaaagctaa cttgtttatt
     3001 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     3061 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     3121 atccgctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     3181 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     3241 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     3301 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     3361 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     3421 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     3481 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     3541 gcgtggcgct ttctcaatgc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     3601 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     3661 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     3721 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     3781 ctaactacgg ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta
     3841 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     3901 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     3961 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     4021 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     4081 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     4141 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     4201 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     4261 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     4321 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     4381 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     4441 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     4501 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     4561 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     4621 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     4681 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     4741 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     4801 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     4861 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     4921 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     4981 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     5041 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     5101 aagtgccacc tg
//
