LOCUS       sequence-343519-        6148 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Conditional express shRNA with control of LoxP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6148)
  AUTHORS   Sun W, Dong H, Balaz M, Slyper M, Drokhlyansky E, Colleluori G, 
            Giordano A, Kovanicova Z, Stefanicka P, Balazova L, Ding L, Husted 
            AS, Rudofsky G, Ukropec J, Cinti S, Schwartz TW, Regev A, Wolfrum C
  TITLE     snRNA-seq reveals a subpopulation of adipocytes that regulates 
            thermogenesis.
  JOURNAL   Nature. 2020 Nov;587(7832):98-102. doi: 10.1038/s41586-020-2856-x. 
            Epub 2020 Oct 28.
  PUBMED    33116305
REFERENCE   2  (bases 1 to 6148)
  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"; doi: 
            "10.1038/s41586-020-2856-x"; journalName: "Nature"; date: "2020-11";
            volume: "587"; issue: "7832"; pages: "98-102"
COMMENT     Sequence Label: AAV-EFS-LSL-nlsGFP-shRNA
FEATURES             Location/Qualifiers
     source          1..6148
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..141
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   12..141
                     /label=AAV2 ITR
     protein_bind    148..168
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        188..399
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     protein_bind    400..424
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     434..450
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     434..450
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     protein_bind    complement(464..497)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    670..804
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    924..1058
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     polyA_signal    1178..1312
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(1341..1374)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    complement(1419..1443)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             1452..1472
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(1473..1475,1476..1478,1479..2189)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1473..1475,1476..1478,1479..2189)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1518..1539)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1779..1798)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2126..2147
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             2190..2216
                     /codon_start=1
                     /product="nuclear localization signal of human c-Myc 
                     proto-oncogene (Dang and Lee, 1988)"
                     /label=c-myc NLS
                     /translation="PAAKRVKLD"
     ncRNA           2372..2386
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     protein_bind    complement(2541..2561)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     misc_feature    2572..3160
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2625..2645)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3043..3054)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(3190..3207)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3196..3403
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3411..3551
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3411..3540
                     /label=AAV2 ITR
     rep_origin      3626..4081
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3713..3732)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3923..3944
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4098..4117)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4217..4239
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4277..4295)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4363..4467
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4468..4536,4537..5328)
                     /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(4686..4705)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5499..6087
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5988..6007
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 cctgcaggca gctgcgcgct cgctcgctca ctgaggccgc ccgggcaaag cccgggcgtc
       61 gggcgacctt tggtcgcccg gcctcagtga gcgagcgagc gcgcagagag ggagtggcca
      121 actccatcac taggggttcc ttctagacaa ctttgtatag aaaagttggg ctccggtgcc
      181 cgtcagtggg cagagcgcac atcgcccaca gtccccgaga agttgggggg aggggtcggc
      241 aattgatccg gtgcctagag aaggtggcgc ggggtaaact gggaaagtga tgtcgtgtac
      301 tggctccgcc tttttcccga gggtggggga gaaccgtata taagtgcagt agtcgccgtg
      361 aacgttcttt ttcgcaacgg gtttgccgcc agaacacagg caagtttgta caaaaaagca
      421 ggctcgaggc cgctctagaa ctagtggatc cggaaccctt aatataactt cgtatagcat
      481 acattatacg aagttattag gtccctcgac ctgcaggaat tagcttgcag atctgcgact
      541 ctagaggatc tgcgactcta gaggatcata atcagccata ccacatttgt agaggtttta
      601 cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat gaatgcaatt
      661 gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
      721 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
      781 aatgtatctt atcatgtctg gatctgcgac tctagaggat cataatcagc cataccacat
      841 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
      901 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
      961 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     1021 tgtccaaact catcaatgta tcttatcatg tctggatctg cgactctaga ggatcataat
     1081 cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac acctccccct
     1141 gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg cagcttataa
     1201 tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt tttcactgca
     1261 ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga tccccatcaa
     1321 gctgatccgg aacccttaat ataacttcgt atagcataca ttatacgaag ttattaggtc
     1381 cctcgacctg cagcccaagc tagcttatcg ataccgtcac ccagctttct tgtacaaagt
     1441 gggccaccat gccaaaaaag aagagaaagg taatggtgag caagggcgag gagctgttca
     1501 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg
     1561 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca
     1621 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacc tacggcgtgc
     1681 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc
     1741 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc
     1801 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg
     1861 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca
     1921 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttc aagatccgcc
     1981 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg
     2041 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca
     2101 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga
     2161 tcactctcgg catggacgag ctgtacaagc ctgctgccaa gagggtcaag ttggactagt
     2221 gtttgaatga ggcttcagta ctttacagaa tcgttgcctg cacatcttgg aaacacttgc
     2281 tgggattact tcgacttctt aacccaacag aaggctcgag aaggtatatt gctgttgaca
     2341 gtgagcgccc taaggttaag tcgccctcgt agtgaagcca cagatgtacg agggcgactt
     2401 aaccttaggt tgcctactgc ctcggacttc aaggggctag aattcgagca attatcttgt
     2461 ttactaaaac tgaatacctt gctatctctt tgatacattt ttacaaagct gaattaaaat
     2521 ggtataaatt aaatcacttt caactttatt atacatagtt ggaattccga taatcaacct
     2581 ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc tccttttacg
     2641 ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg tatggctttc
     2701 attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt gtggcccgtt
     2761 gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac tggttggggc
     2821 attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc tattgccacg
     2881 gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct gttgggcact
     2941 gacaattccg tggtgttgtc ggggaagctg acgtcctttc catggctgct cgcctgtgtt
     3001 gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct caatccagcg
     3061 gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct tcgccttcgc
     3121 cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgggaatt cctagagctc
     3181 gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     3241 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     3301 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     3361 gcaaggggga ggattgggaa gagaatagca ggcatgctgg ggagggccgc aggaacccct
     3421 agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc
     3481 aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag
     3541 ctgcctgcag gggcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
     3601 ccgcatacgt caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt
     3661 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccttagcgcc cgctcctttc
     3721 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3781 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     3841 ttgggtgatg gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg
     3901 ttggagtcca cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaactct
     3961 atctcgggct attcttttga tttataaggg attttgccga tttcggtcta ttggttaaaa
     4021 aatgagctga tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt
     4081 ttatggtgca ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac
     4141 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     4201 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     4261 cgcgcgagac gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata
     4321 atggtttctt agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt
     4381 ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg
     4441 cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt
     4501 cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta
     4561 aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc
     4621 ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa
     4681 gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc
     4741 cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt
     4801 acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact
     4861 gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac
     4921 aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata
     4981 ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta
     5041 ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg
     5101 gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat
     5161 aaatctggag ccggtgagcg tggaagccgc ggtatcattg cagcactggg gccagatggt
     5221 aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga
     5281 aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa
     5341 gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag
     5401 gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5461 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     5521 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     5581 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     5641 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     5701 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     5761 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     5821 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     5881 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     5941 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6001 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     6061 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     6121 gccttttgct ggccttttgc tcacatgt
//
