LOCUS       sequence-382091-        6541 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses 3 non-targeting miRNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6541)
  TITLE     GeneDetect Michael J Fox Foundation Shared Tools Plasmids 2022
REFERENCE   2  (bases 1 to 6541)
  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"
COMMENT     Sequence Label: pAM/CBA-eGFP-miRNegativex3-WPRE-bGHpA
FEATURES             Location/Qualifiers
     source          1..6541
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   4..133
                     /label=AAV2 ITR
     promoter        439..716
                     /label=chicken beta-actin promoter
     primer_bind     995..1017
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1079..1098
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             join(1131..1133,1134..1136,1137..1850)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1131..1133,1134..1136,1137..1847)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1176..1197)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1437..1456)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1784..1805
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     ncRNA           1919..1945
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           2009..2050
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           2057..2083
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           2147..2188
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           2195..2221
                     /label=5' miR-155
                     /note="sequence upstream of the precursor of mouse miR-155 
                     microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     ncRNA           2285..2326
                     /label=3' miR-155
                     /note="sequence downstream of the precursor of mouse 
                     miR-155 microRNA (Uva et al., 2013)"
                     /ncRNA_class="miRNA"
     misc_feature    2364..2952
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2417..2437)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2835..2846)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2979..2996)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2985..3209
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     promoter        complement(3419..3748)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3433..3568
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3733..3753
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(3994..4582)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4074..4093)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4753..5544,5545..5613))
                     /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     5376..5395
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5614..5718)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5786..5804
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(5842..5864)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     5964..5983
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6197..6216
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        6506..6524
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     6506..6523
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 tagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
       61 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
      121 actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc
      181 tctaggtacc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      241 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      301 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      361 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      421 tcatcgctat taccatggtc gaggtgagcc ccacgttctg cttcactctc cccatctccc
      481 ccccctcccc acccccaatt ttgtatttat ttatttttta attattttgt gcagcgatgg
      541 gggcgggggg gggggggggg cgcgcgccag gsggggsggg gsggggsgag gggsggggsg
      601 gggsgaggsg gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt
      661 ttatggcgag gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag
      721 tcgctgcgcg ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc
      781 ggctctgact gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg
      841 gctgtaatta gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc
      901 ttgaggggct ccgggagggc cctttgtgcg gggggagcgg ctcggggctg tccgcggggg
      961 gacggctgcc ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc
     1021 ggctctagag cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc
     1081 aacgtgctgg ttattgtgct gtctcatcat tttggcaaag aattggatcc atggtgagca
     1141 agggcgagga gctgttcacc ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa
     1201 acggccacaa gttcagcgtg tccggcgagg gcgagggcga tgccacctac ggcaagctga
     1261 ccctgaagtt catctgcacc accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca
     1321 ccctgaccta cggcgtgcag tgcttcagcc gctaccccga ccacatgaag cagcacgact
     1381 tcttcaagtc cgccatgccc gaaggctacg tccaggagcg caccatcttc ttcaaggacg
     1441 acggcaacta caagacccgc gccgaggtga agttcgaggg cgacaccctg gtgaaccgca
     1501 tcgagctgaa gggcatcgac ttcaaggagg acggcaacat cctggggcac aagctggagt
     1561 acaactacaa cagccacaac gtctatatca tggccgacaa gcagaagaac ggcatcaagg
     1621 tgaacttcaa gatccgccac aacatcgagg acggcagcgt gcagctcgcc gaccactacc
     1681 agcagaacac ccccatcggc gacggccccg tgctgctgcc cgacaaccac tacctgagca
     1741 cccagtccgc cctgagcaaa gaccccaacg agaagcgcga tcacatggtc ctgctggagt
     1801 tcgtgaccgc cgccgggatc actctcggca tggacgagct gtacaagtaa agcggcccta
     1861 gaggggctgc aggaattgga gtcgaccagt ggatctggcc gcactcgacc agtggatcct
     1921 ggaggcttgc tgaaggctgt atgctgaaat gtactgcgcg tggagacgtt ttggccactg
     1981 actgacgtct ccacgcagta catttcagga cacaaggcct gttactagca ctcacatgga
     2041 acaaatggcc cagatcctgg aggcttgctg aaggctgtat gctgaaatgt actgcgcgtg
     2101 gagacgtttt ggccactgac tgacgtctcc acgcagtaca tttcaggaca caaggcctgt
     2161 tactagcact cacatggaac aaatggccca gatcctggag gcttgctgaa ggctgtatgc
     2221 tgaaatgtac tgcgcgtgga gacgttttgg ccactgactg acgtctccac gcagtacatt
     2281 tcaggacaca aggcctgtta ctagcactca catggaacaa atggcccaga tctggccgca
     2341 ctcgagatat caagcttatc gataatcaac ctctggatta caaaatttgt gaaagattga
     2401 ctggtattct taactatgtt gctcctttta cgctatgtgg atacgctgct ttaatgcctt
     2461 tgtatcatgc tattgcttcc cgtatggctt tcattttctc ctccttgtat aaatcctggt
     2521 tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca acgtggcgtg gtgtgcactg
     2581 tgtttgctga cgcaaccccc actggttggg gcattgccac cacctgtcag ctcctttccg
     2641 ggactttcgc tttccccctc cctattgcca cggcggaact catcgccgcc tgccttgccc
     2701 gctgctggac aggggctcgg ctgttgggca ctgacaattc cgtggtgttg tcggggaaat
     2761 catcgtcctt tccttggctg ctcgcctgtg ttgccacctg gattctgcgc gggacgtcct
     2821 tctgctacgt cccttcggcc ctcaatccag cggaccttcc ttcccgcggc ctgctgccgg
     2881 ctctgcggcc tcttccgcgt cttcgccttc gccctcagac gagtcggatc tccctttggg
     2941 ccgcctcccc gcatcgatac cgtcgactcg ctgatcagcc tcgactgtgc cttctagttg
     3001 ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag gtgccactcc
     3061 cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta ggtgtcattc
     3121 tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag acaatagcag
     3181 gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc
     3241 gactagagca tggctacgta gataagtagc atggcgggtt aatcattaac tacaaggaac
     3301 ccctagtgat ggagttggcc actccctctc tgcgcgctcg ctcgctcact gaggccgggc
     3361 gaccaaaggt cgcccgacgc ccgggcggcc tcagtgagcg agcgagcgcg cagagctttt
     3421 tgcaaaagcc taggcctcca aaaaagcctc ctcactactt ctggaatagc tcagaggccg
     3481 aggcggcctc ggcctctgca taaataaaaa aaattagtca gccatggggc ggagaatggg
     3541 cggaactggg cggagttagg ggcgggatgg gcggagttag gggcgggact atggttgctg
     3601 actaattgag atgcatgctt tgcatacttc tgcctgctgg ggagcctggg gactttccac
     3661 acctggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct ggggagcctg
     3721 gggactttcc acaccctaac tgacacacat tccacagctg cattaatgaa tcggccaacg
     3781 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     3841 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     3901 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     3961 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     4021 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     4081 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     4141 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     4201 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     4261 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     4321 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     4381 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     4441 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     4501 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     4561 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     4621 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     4681 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     4741 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     4801 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     4861 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     4921 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     4981 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     5041 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     5101 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     5161 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     5221 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     5281 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     5341 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     5401 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     5461 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     5521 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     5581 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     5641 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     5701 acaaataggg gttccgcgca catttccccg aaaagtgcca cctgacgtct aagaaaccat
     5761 tattatcatg acattaacct ataaaaatag gcgtatcacg aggccctttc gtctcgcgcg
     5821 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     5881 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     5941 gtgtcggggc tggcttaact atgcggcatc agagcagatt gtactgagag tgcaccattc
     6001 gacgctctcc cttatgcgac tcctgcatta ggaagcagcc cagtagtagg ttgaggccgt
     6061 tgagcaccgc cgccgcaagg aatggtgcat gcaaggagat ggcgcccaac agtcccccgg
     6121 ccacggggcc tgccaccata cccacgccga aacaagcgct catgagcccg aagtggcgag
     6181 cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca cctgtggcgc
     6241 cggtgatgcc ggccacgatg cgtccggcgt agaggatctg gctagcgatg accctgctga
     6301 ttggttcgct gaccatttcc gggtgcggga cggcgttacc agaaactcag aaggttcgtc
     6361 caaccaaacc gactctgacg gcagtttacg agagagatga tagggtctgc ttcagtaagc
     6421 cagatgctac acaattaggc ttgtacatat tgtcgttaga acgcggctac aattaataca
     6481 taaccttatg tatcatacac atacgattta ggtgacacta tagaatacac ggaattaatt
     6541 c
//
