LOCUS       sequence-319410-        6580 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cdkn3 shRNA in pMKO.1 retroviral vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6580)
  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 6580)
  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-shCdkn3
FEATURES             Location/Qualifiers
     source          1..6580
                     /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        340..669
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      520..655
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             679..1278
                     /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(679..698)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1175..1195
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             1474..1899
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     Moloney murine leukemia virus"
     primer_bind     complement(2002..2024)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     promoter        2165..2494
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2345..2480
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2516..2535
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2616..2633)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2787..3375)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2867..2886)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3546..4337,4338..4406))
                     /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     4169..4188
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4407..4511)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4789..4807
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     enhancer        4907..5286
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        5287..5490
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     5441..5461
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             5491..5666
                     /label=5' LTR (truncated)
                     /note="truncated long terminal repeat from Moloney murine 
                     sarcoma virus"
     misc_feature    5729..5928
                     /label=MMLV Psi
                     /note="packaging signal of Moloney murine leukemia virus 
                     (MMLV)"
     CDS             6129..6545
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     6475..6497
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     6513..6529
                     /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 gcatagacag ccttcgagat gtctcgagac atctcgaagg ctgtctatgt
      301 tttttgaatt cgccagcaca gtggtcgacc ctgtggaatg tgtgtcagtt agggtgtgga
      361 aagtccccag gcttcccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca
      421 accaggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
      481 aattagtcag caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc
      541 agttccgccc attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag
      601 gccgcctcgg cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc
      661 ttttgcaaaa agcttaccat gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac
      721 gacgtcccca gggccgtacg caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc
      781 cacaccgtcg atccggaccg ccacatcgag cgggtcaccg agctgcaaga actcttcctc
      841 acgcgcgtcg ggctcgacat cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg
      901 gtctggacca cgccggagag cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc
      961 atggccgagt tgagcggttc ccggctggcc gcgcagcaac agatggaagg cctcctggcg
     1021 ccgcaccggc ccaaggagcc cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac
     1081 cagggcaagg gtctgggcag cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc
     1141 ggggtgcccg ccttcctgga gacctccgcg ccccgcaacc tccccttcta cgagcggctc
     1201 ggcttcaccg tcaccgccga cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc
     1261 cgcaagcccg gtgcctgacg cccgccccac gacccgcagc gcccgaccga aaggagcgca
     1321 cgaccccatg catcgattag tccaatttgt taaagacagg atatcagtgg tccaggctct
     1381 agttttgact caacaatatc accagctgaa gcctatagag tacgagccat agataaaata
     1441 aaagatttta tttagtctcc agaaaaaggg gggaatgaaa gaccccacct gtaggtttgg
     1501 caagctagct taagtaacgc cattttgcaa ggcatggaaa aatacataac tgagaataga
     1561 gaagttcaga tcaaggtcag gaacagatgg aacagggtcg accctagaga accatcagat
     1621 gtttccaggg tgccccaagg acctgaaatg accctgtgcc ttatttgaac taaccaatca
     1681 gttcgcttct cgcttctgtt cgcgcgcttc tgctccccga gctcaataaa agagcccaca
     1741 acccctcact cggggcgcca gtcctccgat tgactgagtc gcccgggtac ccgtgtatcc
     1801 aataaaccct cttgcagttg catccgactt gtggtctcgc tgttccttgg gagggtctcc
     1861 tctgagtgat tgactacccg tcagcggggg tctttcattt gggggctcgt ccgggatcgg
     1921 gagacccctg cccagggacc accgacccac caccgggagg taagctggct gcctcgcgcg
     1981 tttcggtgat gacggtgaaa acctctgaca catgcagctc ccggagacgg tcacagcttg
     2041 tctgtaagcg gatgccggga gcagacaagc ccgtcagggc gcgtcagcgg gtgttggcgg
     2101 gtgtcggggc gcagccatga cccagtcacg tagcgatagc ggagtgtaga tccggctgtg
     2161 gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca
     2221 aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg
     2281 cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     2341 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     2401 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     2461 aggaggcttt tttggaggcc taggcttttg caaaaagctt actggcttaa ctatgcggca
     2521 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta
     2581 aggagaaaat accgcatcag gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     2641 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     2701 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     2761 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     2821 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     2881 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     2941 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     3001 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     3061 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     3121 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     3181 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     3241 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     3301 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     3361 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     3421 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     3481 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     3541 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     3601 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     3661 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     3721 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     3781 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     3841 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     3901 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     3961 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     4021 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     4081 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     4141 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     4201 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     4261 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     4321 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     4381 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     4441 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4501 ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt
     4561 taaaattcgc gttaaatttt tgttaaatca gctcattttt taaccaatag gccgaaatcg
     4621 gcaaaatccc ttaattattg aagcatttat cagggttatt gtctcatgag cggatacata
     4681 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     4741 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     4801 acgaggccct ttcgtcttca agaattagct tggccattgc atacgttgta tccatatcat
     4861 aatatgtaca tttatattgg ctcatgtcca acattaccgc catgttgaca ttgattattg
     4921 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
     4981 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
     5041 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
     5101 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
     5161 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
     5221 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
     5281 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
     5341 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
     5401 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
     5461 gtacggtggg aggtctatat aagcagagct caataaaaga gcccacaacc cctcactcgg
     5521 cgcgccagtc ttccgataga ctgcgtcgcc cgggtacccg tattcccaat aaagcctctt
     5581 gctgtttgca tccgaatcgt ggtctcgctg ttccttggga gggtctcctc tgagtgattg
     5641 actacccacg acgggggtct ttcatttggg ggctcgtccg ggatttggag acccctgccc
     5701 agggaccacc gacccaccac cgggaggtaa gctggccagc aacttatctg tgtctgtccg
     5761 attgtctagt gtctatgttt gatgttatgc gcctgcgtct gtactagtta gctaactagc
     5821 tctgtatctg gcggacccgt ggtggaactg acgagttctg aacacccggc cgcaaccctg
     5881 ggagacgtcc cagggacttt gggggccgtt tttgtggccc gacctgagga agggagtcga
     5941 tgtggaatcc gaccccgtca ggatatgtgg ttctggtagg agacgagaac ctaaaacagt
     6001 tcccgcctcc gtctgaattt ttgctttcgg tttggaaccg aagccgcgcg tcttgtctgc
     6061 tgcagcgctg cagcatcgtt ctgtgttgtc tctgtctgac tgtgtttctg tatttgtctg
     6121 aaaattaggg ccagactgtt accactccct taagtttgac cttaggtcac tggaaagatg
     6181 tcgagcggat cgctcacaac cagtcggtag atgtcaagaa gagacgttgg gttaccttct
     6241 gctctgcaga atggccaacc tttaacgtcg gatggccgcg agacggcacc tttaaccgag
     6301 acctcatcac ccaggttaag atcaaggtct tttcacctgg cccgcatgga cacccagacc
     6361 aggtccccta catcgtgacc tgggaagcct tggcttttga cccccctccc tgggtcaagc
     6421 cctttgtaca ccctaagcct ccgcctcctc ttcctccatc cgccccgtct ctcccccttg
     6481 aacctcctcg ttcgaccccg cctcgatcct ccctttatcc agccctcact ccttctctag
     6541 gcgccggaat tgaagatctg tcgacaaggt cgggcaggaa
//
