LOCUS       sequence-337927-        8166 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Constitutive overexpression of miRNA or shRNA with mTagBFP2 as 
            reporter fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8166)
  TITLE     Heckl Lab Plasmids
REFERENCE   2  (bases 1 to 8166)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: SIN40C.SFFV.mTagBFP2.miR30n
FEATURES             Location/Qualifiers
     source          1..8166
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    15..603
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(68..88)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(486..497)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             675..908
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    986..1107
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1023..1042)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1077..1096
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1147..1282
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1302..1321)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1303..1321)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1331..1348)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1331..1347)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1340..1362)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1489..1944
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1576..1595)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1786..1807
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1970..2074
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2075..2143,2144..2935)
                     /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(2293..2312)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3106..3694
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3595..3614
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3848..3865
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3982..4003
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4018..4023,4024..4041,4042..4048)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4056..4072
                     /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     4061..4083
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4080..4096
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4080..4096
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4115..4135
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4117..4135
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     enhancer        4180..4559
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     enhancer        4616..4807
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4639..4659)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4826..5052
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5053..5233
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5280..5405
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5898..6131
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6316..6360
                     /codon_start=1
                     /product="antigenic peptide corresponding to amino acids 
                     655 to 669 of the HIV envelope protein gp41 (Lutje Hulsik 
                     et al., 2013)"
                     /label=gp41 peptide
                     /note="recognized by the 2H10 single-chain llama nanobody"
                     /translation="KNEQELLELDKWASL"
     misc_feature    6573..6690
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6748..7155
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      7155..7164
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     ncRNA           7887..8008
                     /label=5' miR-30a
                     /note="sequence upstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     ncRNA           8036..8162
                     /label=3' miR-30a
                     /note="sequence downstream of the 71-nt precursor of the 
                     human miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
ORIGIN
        1 cgcgttaagt cgacaatcaa cctctggatt acaaaatttg tgaaagattg actggtattc
       61 ttaactatgt tgctcctttt acgctatgtg gatacgctgc tttaatgcct ttgtatcatg
      121 ctattgcttc ccgtatggct ttcattttct cctccttgta taaatcctgg ttgctgtctc
      181 tttatgagga gttgtggccc gttgtcaggc aacgtggcgt ggtgtgcact gtgtttgctg
      241 acgcaacccc cactggttgg ggcattgcca ccacctgtca gctcctttcc gggactttcg
      301 ctttccccct ccctattgcc acggcggaac tcatcgccgc ctgccttgcc cgctgctgga
      361 caggggctcg gctgttgggc actgacaatt ccgtggtgtt gtcggggaaa tcatcgtcct
      421 ttccttggct gctcgcctgt gttgccacct ggattctgcg cgggacgtcc ttctgctacg
      481 tcccttcggc cctcaatcca gcggaccttc cttcccgcgg cctgctgccg gctctgcggc
      541 ctcttccgcg tcttcgcctt cgccctcaga cgagtcggat ctccctttgg gccgcctccc
      601 cgcgtcgact ttaagaccaa tgacttacaa ggcagctgta gatcttagcc actttttaaa
      661 agaaaagggg ggactggaag ggctaattca ctcccaacga agacaagatc tgctttttgc
      721 ttgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
      781 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
      841 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
      901 ctctagcagt acgtatagta gttcatgtca tcttattatt cagtatttat aacttgcaaa
      961 gaaatgaata tcagagagtg agaggaactt gtttattgca gcttataatg gttacaaata
     1021 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     1081 tttgtccaaa ctcatcaatg tatcttatca tgtctggctc tagctatccc gcccctaact
     1141 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta
     1201 atttttttta tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag
     1261 tgaggaggct tttttggagg cctagggacg tacccaattc gccctatagt gagtcgtatt
     1321 acgcgcgctc actggccgtc gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc
     1381 aacttaatcg ccttgcagca catccccctt tcgccagctg gcgtaatagc gaagaggccc
     1441 gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg cgaatgggac gcgccctgta
     1501 gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca
     1561 gcgccctagc gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct
     1621 ttccccgtca agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc
     1681 acctcgaccc caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat
     1741 agacggtttt tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc
     1801 aaactggaac aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc
     1861 cgatttcggc ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta
     1921 acaaaatatt aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     1981 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2041 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     2101 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     2161 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     2221 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     2281 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     2341 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     2401 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     2461 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     2521 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     2581 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     2641 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     2701 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     2761 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     2821 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     2881 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     2941 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     3001 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     3061 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3121 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3181 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3241 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3301 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3361 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     3421 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     3481 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     3541 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     3601 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     3661 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     3721 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     3781 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     3841 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     3901 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     3961 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     4021 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     4081 aggaaacagc tatgaccatg attacgccaa gcgcgcaatt aaccctcact aaagggaaca
     4141 aaagctggag ctgcaagcgg taccgtcgac ctgcaggttg acattgatta ttgactagtt
     4201 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     4261 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     4321 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     4381 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagta
     4441 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     4501 ccttatggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgg
     4561 cgcgccatgc agggcctgaa ataacctctg aaagaggaac ttggttaggt accttctgag
     4621 gcggaaagaa ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc
     4681 cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt
     4741 ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca
     4801 tagtccctta agaggcgcgc cttaatgtag tcttatgcaa tactcttgta gtcttgcaac
     4861 atggtaacga tgagttagca acatgcctta caaggagaga aaaagcaccg tgcatgccga
     4921 ttggtggaag taaggtggta cgatcgtgcc ttattaggaa ggcaacagac gggtctgaca
     4981 tggattggac gaaccactga attgccgcat tgcagagata ttgtatttaa gtgcctagct
     5041 cgatacataa acgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa
     5101 ctagggaacc cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt
     5161 gcccgtctgt tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg
     5221 aaaatctcta gcagtggcgc ccgaacaggg acttgaaagc gaaagggaaa ccagaggagc
     5281 tctctcgacg caggactcgg cttgctgaag cgcgcacggc aagaggcgag gggcggcgac
     5341 tggtgagtac gccaaaaatt ttgactagcg gaggctagaa ggagagagat gggtgcgaga
     5401 gcgtcagtat taagcggggg agaattagat cgcgatggga aaaaattcgg ttaaggccag
     5461 ggggaaagaa aaaatataaa ttaaaacata tagtatgggc aagcagggag ctagaacgat
     5521 tcgcagttaa tcctggcctg ttagaaacat cagaaggctg tagacaaata ctgggacagc
     5581 tacaaccatc ccttcagaca ggatcagaag aacttagatc attatataat acagtagcaa
     5641 ccctctattg tgtgcatcaa aggatagaga taaaagacac caaggaagct ttagacaaga
     5701 tagaggaaga gcaaaacaaa agtaagacca ccgcacagca agcggccgct gatcttcaga
     5761 cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata taaagtagta
     5821 aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt gcagagagaa
     5881 aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc aggaagcact
     5941 atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc tggtatagtg
     6001 cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt gcaactcaca
     6061 gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata cctaaaggat
     6121 caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac tgctgtgcct
     6181 tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac gacctggatg
     6241 gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat tgaagaatcg
     6301 caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg ggcaagtttg
     6361 tggaattggt ttaacataac aaattggctg tggtatataa aattattcat aatgatagta
     6421 ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa tagagttagg
     6481 cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg acaattctag
     6541 atcttgagac aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg
     6601 tacagtgcag gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta
     6661 caaaaacaaa ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca
     6721 ctttggcgcc ggctcgagcg agctgcagta acgccatttt gcaaggcatg gaaaaatacc
     6781 aaaccaagaa tagagaagtt cagatcaagg gcgggtacat gaaaatagct aacgttgggc
     6841 caaacaggat atctgcggtg agcagtttcg gccccggccc ggggccaaga acagatggtc
     6901 accgcagttt cggccccggc ccgaggccaa gaacagatgg tccccagata tggcccaacc
     6961 ctcagcagtt tcttaagacc catcagatgt ttccaggctc ccccaaggac ctgaaatgac
     7021 cctgcgcctt atttgaatta accaatcagc ctgcttctcg cttctgttcg cgcgcttctg
     7081 cttcccgagc tctataaaag agctcacaac ccctcactcg gcgcgccagt cctccgacag
     7141 actgagtcgg atccgccacc atgggaagcg agctgattaa ggagaacatg cacatgaagc
     7201 tctacatgga gggcaccgtg gacaaccatc acttcaagtg cacatccgag ggcgaaggca
     7261 agccctacga gggcacccag accatgagaa tcaaggtggt cgagggcggc cctctcccct
     7321 tcgccttcga catcctggct actagcttcc tctacggcag caagaccttc atcaaccaca
     7381 cccagggcat ccccgacttc ttcaagcagt ccttccctga gggcttcaca tgggagagag
     7441 tcaccacata cgaagacggg ggcgtgctga ccgctaccca ggacaccagc ctccaggacg
     7501 gctgcctcat ctacaacgtc aagatcagag gggtgaactt cacatccaac ggccctgtga
     7561 tgcagaagaa aacactcggc tgggaggcct tcaccgagac cctgtacccc gctgacggcg
     7621 gcctggaagg cagaaacgac atggccctga agctcgtggg cgggagccat ctgatcgcaa
     7681 acatcaagac cacatataga tccaagaaac ccgctaagaa cctcaagatg cctggcgtct
     7741 actatgtgga ctacagactg gaaagaatca aggaggccaa caacgagacc tacgtcgagc
     7801 agcacgaggt ggcagtggcc agatactgcg acctccctag caaactgggg cacaagctgt
     7861 acaagtgagc ggccgcatat tgctgtttga atgaggcttc agtactttac agaatcgttg
     7921 cctgcacatc ttggaaacac ttgctgggat tacttcttca ggttaaccca acagaaggct
     7981 aaagaaggta tattgctgtt gacagtgagc gggagacgtg attaccgtct cttgcctact
     8041 gcctcggact tcaaggggct actttaggag caattatctt gtttactaaa actgaatacc
     8101 ttgctatctc tttgatacat ttttacaaag ctgaattaaa atggtataaa ttaaatcact
     8161 ttttta
//
