LOCUS       sequence-319413-        6579 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Spc25 shRNA in pMKO.1 retroviral vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6579)
  AUTHORS   Zhang Y, Federation AJ, Kim S, O'Keefe JP, Lun M, Xiang D, Brown JD,
            Steinhauser ML
  TITLE     Targeting nuclear receptor NR4A1-dependent adipocyte progenitor 
            quiescence promotes metabolic adaptation to obesity.
  JOURNAL   J Clin Invest. 2018 Nov 1;128(11):4898-4911. doi: 10.1172/JCI98353. 
            Epub 2018 Oct 2.
  PUBMED    30277475
REFERENCE   2  (bases 1 to 6579)
  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: "10.1172/JCI98353"; 
            journalName: "J Clin Invest"; date: "2018-11-1- 1"; volume: "128"; 
            issue: "11"; pages: "4898-4911"
COMMENT     Sequence Label: pMKO.1-shSpc25
FEATURES             Location/Qualifiers
     source          1..6579
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        339..668
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      519..654
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             678..1277
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(678..697)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1174..1194
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1473..1898
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(2001..2023)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        2164..2493
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2344..2479
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2515..2534
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2615..2632)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2786..3374)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2866..2885)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3545..4336,4337..4405))
                     /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     4168..4187
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4406..4510)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4788..4806
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        4906..5285
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5286..5489
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5440..5460
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             5490..5665
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    5728..5927
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6128..6544
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6474..6496
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6512..6528
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattagaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg gccagctctg taagcagaat aactcgagtt attctgctta cagagctggt
      301 ttttgaattc gccagcacag tggtcgaccc tgtggaatgt gtgtcagtta gggtgtggaa
      361 agtccccagg cttcccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
      421 ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca
      481 attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca
      541 gttccgccca ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg
      601 ccgcctcggc ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct
      661 tttgcaaaaa gcttaccatg accgagtaca agcccacggt gcgcctcgcc acccgcgacg
      721 acgtccccag ggccgtacgc accctcgccg ccgcgttcgc cgactacccc gccacgcgcc
      781 acaccgtcga tccggaccgc cacatcgagc gggtcaccga gctgcaagaa ctcttcctca
      841 cgcgcgtcgg gctcgacatc ggcaaggtgt gggtcgcgga cgacggcgcc gcggtggcgg
      901 tctggaccac gccggagagc gtcgaagcgg gggcggtgtt cgccgagatc ggcccgcgca
      961 tggccgagtt gagcggttcc cggctggccg cgcagcaaca gatggaaggc ctcctggcgc
     1021 cgcaccggcc caaggagccc gcgtggttcc tggccaccgt cggcgtctcg cccgaccacc
     1081 agggcaaggg tctgggcagc gccgtcgtgc tccccggagt ggaggcggcc gagcgcgccg
     1141 gggtgcccgc cttcctggag acctccgcgc cccgcaacct ccccttctac gagcggctcg
     1201 gcttcaccgt caccgccgac gtcgaggtgc ccgaaggacc gcgcacctgg tgcatgaccc
     1261 gcaagcccgg tgcctgacgc ccgccccacg acccgcagcg cccgaccgaa aggagcgcac
     1321 gaccccatgc atcgattagt ccaatttgtt aaagacagga tatcagtggt ccaggctcta
     1381 gttttgactc aacaatatca ccagctgaag cctatagagt acgagccata gataaaataa
     1441 aagattttat ttagtctcca gaaaaagggg ggaatgaaag accccacctg taggtttggc
     1501 aagctagctt aagtaacgcc attttgcaag gcatggaaaa atacataact gagaatagag
     1561 aagttcagat caaggtcagg aacagatgga acagggtcga ccctagagaa ccatcagatg
     1621 tttccagggt gccccaagga cctgaaatga ccctgtgcct tatttgaact aaccaatcag
     1681 ttcgcttctc gcttctgttc gcgcgcttct gctccccgag ctcaataaaa gagcccacaa
     1741 cccctcactc ggggcgccag tcctccgatt gactgagtcg cccgggtacc cgtgtatcca
     1801 ataaaccctc ttgcagttgc atccgacttg tggtctcgct gttccttggg agggtctcct
     1861 ctgagtgatt gactacccgt cagcgggggt ctttcatttg ggggctcgtc cgggatcggg
     1921 agacccctgc ccagggacca ccgacccacc accgggaggt aagctggctg cctcgcgcgt
     1981 ttcggtgatg acggtgaaaa cctctgacac atgcagctcc cggagacggt cacagcttgt
     2041 ctgtaagcgg atgccgggag cagacaagcc cgtcagggcg cgtcagcggg tgttggcggg
     2101 tgtcggggcg cagccatgac ccagtcacgt agcgatagcg gagtgtagat ccggctgtgg
     2161 aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     2221 agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc cccagcaggc
     2281 agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc cctaactccg
     2341 cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt
     2401 ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga
     2461 ggaggctttt ttggaggcct aggcttttgc aaaaagctta ctggcttaac tatgcggcat
     2521 cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac agatgcgtaa
     2581 ggagaaaata ccgcatcagg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
     2641 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     2701 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     2761 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     2821 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     2881 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     2941 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     3001 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     3061 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     3121 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     3181 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     3241 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     3301 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     3361 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     3421 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     3481 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     3541 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     3601 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     3661 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     3721 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     3781 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     3841 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     3901 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     3961 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     4021 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     4081 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     4141 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     4201 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     4261 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     4321 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     4381 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     4441 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     4501 gggttccgcg cacatttccc cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt
     4561 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     4621 caaaatccct taattattga agcatttatc agggttattg tctcatgagc ggatacatat
     4681 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     4741 cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat aggcgtatca
     4801 cgaggccctt tcgtcttcaa gaattagctt ggccattgca tacgttgtat ccatatcata
     4861 atatgtacat ttatattggc tcatgtccaa cattaccgcc atgttgacat tgattattga
     4921 ctagttatta atagtaatca attacggggt cattagttca tagcccatat atggagttcc
     4981 gcgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat
     5041 tgacgtcaat aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc
     5101 aatgggtgga gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc
     5161 caagtacgcc ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt
     5221 acatgacctt atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta
     5281 ccatggtgat gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg
     5341 gatttccaag tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac
     5401 gggactttcc aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg
     5461 tacggtggga ggtctatata agcagagctc aataaaagag cccacaaccc ctcactcggc
     5521 gcgccagtct tccgatagac tgcgtcgccc gggtacccgt attcccaata aagcctcttg
     5581 ctgtttgcat ccgaatcgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga
     5641 ctacccacga cgggggtctt tcatttgggg gctcgtccgg gatttggaga cccctgccca
     5701 gggaccaccg acccaccacc gggaggtaag ctggccagca acttatctgt gtctgtccga
     5761 ttgtctagtg tctatgtttg atgttatgcg cctgcgtctg tactagttag ctaactagct
     5821 ctgtatctgg cggacccgtg gtggaactga cgagttctga acacccggcc gcaaccctgg
     5881 gagacgtccc agggactttg ggggccgttt ttgtggcccg acctgaggaa gggagtcgat
     5941 gtggaatccg accccgtcag gatatgtggt tctggtagga gacgagaacc taaaacagtt
     6001 cccgcctccg tctgaatttt tgctttcggt ttggaaccga agccgcgcgt cttgtctgct
     6061 gcagcgctgc agcatcgttc tgtgttgtct ctgtctgact gtgtttctgt atttgtctga
     6121 aaattagggc cagactgtta ccactccctt aagtttgacc ttaggtcact ggaaagatgt
     6181 cgagcggatc gctcacaacc agtcggtaga tgtcaagaag agacgttggg ttaccttctg
     6241 ctctgcagaa tggccaacct ttaacgtcgg atggccgcga gacggcacct ttaaccgaga
     6301 cctcatcacc caggttaaga tcaaggtctt ttcacctggc ccgcatggac acccagacca
     6361 ggtcccctac atcgtgacct gggaagcctt ggcttttgac ccccctccct gggtcaagcc
     6421 ctttgtacac cctaagcctc cgcctcctct tcctccatcc gccccgtctc tcccccttga
     6481 acctcctcgt tcgaccccgc ctcgatcctc cctttatcca gccctcactc cttctctagg
     6541 cgccggaatt gaagatctgt cgacaaggtc gggcaggaa
//
