LOCUS       sequence-337932-        8163 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive overexpression of miRNA or shRNA with dTomato as 
            reporter fluorescent protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8163)
  TITLE     Heckl Lab Plasmids
REFERENCE   2  (bases 1 to 8163)
  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: SIN40C.SFFV.dTomato.miR30n
FEATURES             Location/Qualifiers
     source          1..8163
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    10..598
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(63..83)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(481..492)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     LTR             670..903
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    981..1102
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1018..1037)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1072..1091
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      1142..1277
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1297..1316)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1298..1316)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1326..1343)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1326..1342)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1335..1357)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      1484..1939
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1571..1590)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1781..1802
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        1965..2069
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2070..2138,2139..2930)
                     /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(2288..2307)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3101..3689
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3590..3609
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3843..3860
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3977..3998
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4013..4018,4019..4036,4037..4043)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4051..4067
                     /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     4056..4078
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4075..4091
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4075..4091
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     4110..4130
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        4112..4130
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     enhancer        4175..4554
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     enhancer        4611..4802
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4634..4654)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4821..5047
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             5048..5228
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    5275..5400
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    5893..6126
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             6311..6355
                     /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    6568..6685
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        6743..7150
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      7150..7159
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             7156..7857
                     /codon_start=1
                     /product="dimeric variant of DsRed fluorescent protein 
                     (Shaner et al., 2004)"
                     /label=dTomato
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEVIKEFMRFKVRMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYKKLSFPEGFKWERVMNFEDGG
                     LVTVTQDSSLQDGTLIYKVKMRGTNFPPDGPVMQKKTMGWEASTERLYPRDGVLKGEIH
                     QALKLKDGGHYLVEFKTIYMAKKPVQLPGYYYVDTKLDITSHNEDYTIVEQYERSEGRH
                     HLFLYGMDELYK"
     primer_bind     complement(7289..7307)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     7557..7576
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     7751..7774
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     ncRNA           7879..8000
                     /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           8028..8154
                     /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 taagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
       61 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgctatt
      121 gcttcccgta tggctttcat tttctcctcc ttgtataaat cctggttgct gtctctttat
      181 gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt gcactgtgtt tgctgacgca
      241 acccccactg gttggggcat tgccaccacc tgtcagctcc tttccgggac tttcgctttc
      301 cccctcccta ttgccacggc ggaactcatc gccgcctgcc ttgcccgctg ctggacaggg
      361 gctcggctgt tgggcactga caattccgtg gtgttgtcgg ggaaatcatc gtcctttcct
      421 tggctgctcg cctgtgttgc cacctggatt ctgcgcggga cgtccttctg ctacgtccct
      481 tcggccctca atccagcgga ccttccttcc cgcggcctgc tgccggctct gcggcctctt
      541 ccgcgtcttc gccttcgccc tcagacgagt cggatctccc tttgggccgc ctccccgcgt
      601 cgactttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
      661 aggggggact ggaagggcta attcactccc aacgaagaca agatctgctt tttgcttgta
      721 ctgggtctct ctggttagac cagatctgag cctgggagct ctctggctaa ctagggaacc
      781 cactgcttaa gcctcaataa agcttgcctt gagtgcttca agtagtgtgt gcccgtctgt
      841 tgtgtgactc tggtaactag agatccctca gaccctttta gtcagtgtgg aaaatctcta
      901 gcagtacgta tagtagttca tgtcatctta ttattcagta tttataactt gcaaagaaat
      961 gaatatcaga gagtgagagg aacttgttta ttgcagctta taatggttac aaataaagca
     1021 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     1081 ccaaactcat caatgtatct tatcatgtct ggctctagct atcccgcccc taactccgcc
     1141 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     1201 ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga agtagtgagg
     1261 aggctttttt ggaggcctag ggacgtaccc aattcgccct atagtgagtc gtattacgcg
     1321 cgctcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt tacccaactt
     1381 aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga ggcccgcacc
     1441 gatcgccctt cccaacagtt gcgcagcctg aatggcgaat gggacgcgcc ctgtagcggc
     1501 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     1561 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     1621 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     1681 gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     1741 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     1801 ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat tttgccgatt
     1861 tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     1921 atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1981 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     2041 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     2101 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     2161 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     2221 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     2281 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     2341 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     2401 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     2461 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     2521 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     2581 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     2641 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     2701 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     2761 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     2821 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     2881 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     2941 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     3001 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     3061 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     3121 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     3181 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     3241 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     3301 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     3361 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3421 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3481 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3541 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3601 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3661 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3721 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3781 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3841 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     3901 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     3961 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     4021 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     4081 acagctatga ccatgattac gccaagcgcg caattaaccc tcactaaagg gaacaaaagc
     4141 tggagctgca agcggtaccg tcgacctgca ggttgacatt gattattgac tagttattaa
     4201 tagtaatcaa ttacggggtc attagttcat agcccatata tggagttccg cgttacataa
     4261 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     4321 atgacgtatg ttcccatagt aacgccaata gggactttcc attgacgtca atgggtggag
     4381 tatttacggt aaactgccca cttggcagta catcaagtgt atcatatgcc aagtacgccc
     4441 cctattgacg tcaatgacgg taaatggccc gcctggcatt atgcccagta catgacctta
     4501 tgggactttc ctacttggca gtacatctac gtattagtca tcgctattac catggcgcgc
     4561 catgcagggc ctgaaataac ctctgaaaga ggaacttggt taggtacctt ctgaggcgga
     4621 aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg ctccccagca
     4681 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg aaagtcccca
     4741 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccatagtc
     4801 ccttaagagg cgcgccttaa tgtagtctta tgcaatactc ttgtagtctt gcaacatggt
     4861 aacgatgagt tagcaacatg ccttacaagg agagaaaaag caccgtgcat gccgattggt
     4921 ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa cagacgggtc tgacatggat
     4981 tggacgaacc actgaattgc cgcattgcag agatattgta tttaagtgcc tagctcgata
     5041 cataaacggg tctctctggt tagaccagat ctgagcctgg gagctctctg gctaactagg
     5101 gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag tgtgtgcccg
     5161 tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag tgtggaaaat
     5221 ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag ggaaaccaga ggagctctct
     5281 cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag gcgaggggcg gcgactggtg
     5341 agtacgccaa aaattttgac tagcggaggc tagaaggaga gagatgggtg cgagagcgtc
     5401 agtattaagc gggggagaat tagatcgcga tgggaaaaaa ttcggttaag gccaggggga
     5461 aagaaaaaat ataaattaaa acatatagta tgggcaagca gggagctaga acgattcgca
     5521 gttaatcctg gcctgttaga aacatcagaa ggctgtagac aaatactggg acagctacaa
     5581 ccatcccttc agacaggatc agaagaactt agatcattat ataatacagt agcaaccctc
     5641 tattgtgtgc atcaaaggat agagataaaa gacaccaagg aagctttaga caagatagag
     5701 gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg ccgctgatct tcagacctgg
     5761 aggaggagat atgagggaca attggagaag tgaattatat aaatataaag tagtaaaaat
     5821 tgaaccatta ggagtagcac ccaccaaggc aaagagaaga gtggtgcaga gagaaaaaag
     5881 agcagtggga ataggagctt tgttccttgg gttcttggga gcagcaggaa gcactatggg
     5941 cgcagcgtca atgacgctga cggtacaggc cagacaatta ttgtctggta tagtgcagca
     6001 gcagaacaat ttgctgaggg ctattgaggc gcaacagcat ctgttgcaac tcacagtctg
     6061 gggcatcaag cagctccagg caagaatcct ggctgtggaa agatacctaa aggatcaaca
     6121 gctcctgggg atttggggtt gctctggaaa actcatttgc accactgctg tgccttggaa
     6181 tgctagttgg agtaataaat ctctggaaca gatttggaat cacacgacct ggatggagtg
     6241 ggacagagaa attaacaatt acacaagctt aatacactcc ttaattgaag aatcgcaaaa
     6301 ccagcaagaa aagaatgaac aagaattatt ggaattagat aaatgggcaa gtttgtggaa
     6361 ttggtttaac ataacaaatt ggctgtggta tataaaatta ttcataatga tagtaggagg
     6421 cttggtaggt ttaagaatag tttttgctgt actttctata gtgaatagag ttaggcaggg
     6481 atattcacca ttatcgtttc agacccacct cccaaccccg aggggacaat tctagatctt
     6541 gagacaaatg gcagtattca tccacaattt taaaagaaaa ggggggattg gggggtacag
     6601 tgcaggggaa agaatagtag acataatagc aacagacata caaactaaag aattacaaaa
     6661 acaaattaca aaaattcaaa attttcgggt ttattacagg gacagcagag atccactttg
     6721 gcgccggctc gagcgagctg cagtaacgcc attttgcaag gcatggaaaa ataccaaacc
     6781 aagaatagag aagttcagat caagggcggg tacatgaaaa tagctaacgt tgggccaaac
     6841 aggatatctg cggtgagcag tttcggcccc ggcccggggc caagaacaga tggtcaccgc
     6901 agtttcggcc ccggcccgag gccaagaaca gatggtcccc agatatggcc caaccctcag
     6961 cagtttctta agacccatca gatgtttcca ggctccccca aggacctgaa atgaccctgc
     7021 gccttatttg aattaaccaa tcagcctgct tctcgcttct gttcgcgcgc ttctgcttcc
     7081 cgagctctat aaaagagctc acaacccctc actcggcgcg ccagtcctcc gacagactga
     7141 gtcggatccg ccaccatggt gagcaagggc gaggaggtca tcaaagagtt catgcgcttc
     7201 aaggtgcgca tggagggctc catgaacggc cacgagttcg agatcgaggg cgagggcgag
     7261 ggccgcccct acgagggcac ccagaccgcc aagctgaagg tgaccaaggg cggccccctg
     7321 cccttcgcct gggacatcct gtccccccag ttcatgtacg gctccaaggc gtacgtgaag
     7381 caccccgccg acatccccga ttacaagaag ctgtccttcc ccgagggctt caagtgggag
     7441 cgcgtgatga acttcgagga cggcggtctg gtgaccgtga cccaggactc ctccctgcag
     7501 gacggcacgc tgatctacaa ggtgaagatg cgcggcacca acttcccccc cgacggcccc
     7561 gtaatgcaga agaagaccat gggctgggag gcctccaccg agcgcctgta cccccgcgac
     7621 ggcgtgctga agggcgagat ccaccaggcc ctgaagctga aggacggcgg ccactacctg
     7681 gtggagttca agaccatcta catggccaag aagcccgtgc aactgcccgg ctactactac
     7741 gtggacacca agctggacat cacctcccac aacgaggact acaccatcgt ggaacagtac
     7801 gagcgctccg agggccgcca ccacctgttc ctgtacggca tggacgagct gtacaagtga
     7861 gcggccgcat attgctgttt gaatgaggct tcagtacttt acagaatcgt tgcctgcaca
     7921 tcttggaaac acttgctggg attacttctt caggttaacc caacagaagg ctaaagaagg
     7981 tatattgctg ttgacagtga gcgggagacg tgattaccgt ctcttgccta ctgcctcgga
     8041 cttcaagggg ctactttagg agcaattatc ttgtttacta aaactgaata ccttgctatc
     8101 tctttgatac atttttacaa agctgaatta aaatggtata aattaaatca cttttttacg
     8161 cgt
//
