LOCUS       sequence-472203-        7008 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Retroviral vector for U6 promoter driven expression empty miR30 
            based shRNA (to be used in conjunction with Phoenix packaging 
            cells)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7008)
  AUTHORS   Hoare M, Ito Y, Kang TW, Weekes MP, Matheson NJ, Patten DA, Shetty 
            S, Parry AJ, Menon S, Salama R, Antrobus R, Tomimatsu K, Howat W, 
            Lehner PJ, Zender L, Narita M
  TITLE     NOTCH1 mediates a switch between two distinct secretomes during 
            senescence.
  JOURNAL   Nat Cell Biol. 2016 Sep;18(9):979-92. doi: 10.1038/ncb3397. Epub 
            2016 Aug 15.
  PUBMED    27525720
REFERENCE   2  (bases 1 to 7008)
  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/ncb3397"; 
            journalName: "Nat Cell Biol"; date: "2016-09"; volume: "18"; issue: 
            "9"; pages: "979-92"
COMMENT     Sequence Label: pMSCV-miR30-Vector
FEATURES             Location/Qualifiers
     source          1..7008
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    579..920
                     /label=MESV Psi
                     /note="packaging signal of murine embryonic stem cell 
                     virus"
     CDS             987..1403
                     /label=gag (truncated)
                     /note="truncated Moloney murine leukemia virus (MMLV) gag 
                     gene lacking the start codon"
     primer_bind     1333..1355
                     /label=pLXSN 5'
                     /note="Murine stem cell virus, forward primer. Also called 
                     MSCV"
     primer_bind     1371..1387
                     /label=pBABE 5'
                     /note="Psi packaging signal, forward primer for pBABE 
                     vectors"
     promoter        1646..1894
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1646..1666
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1817..1836
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     ncRNA           1949..2042
                     /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           2056..2168
                     /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"
     promoter        2182..2681
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2217..2240)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2614..2633
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2702..3301
                     /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(2702..2721)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3198..3218
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3401..3915
                     /label=3' LTR
                     /note="3' long terminal repeat from murine embryonic stem 
                     cell virus"
     protein_bind    4077..4093
                     /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)."
     promoter        complement(join(4101..4107,4108..4125,4126..4131))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4146..4167
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4284..4301)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4455..5043)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4535..4554)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5214..6005,6006..6074))
                     /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     5837..5856
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6075..6179)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6247..6265
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6303..6325)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6425..6444
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6638..6660
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6788..6807
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     LTR             join(7007..7008,1..515)
                     /label=5' LTR
                     /note="5' long terminal repeat from murine embryonic stem 
                     cell virus"
ORIGIN
        1 tgaaagaccc cacctgtagg tttggcaagc tagcttaagt aacgccattt tgcaaggcat
       61 ggaaaataca taactgagaa tagagaagtt cagatcaagg ttaggaacag agagacagca
      121 gaatatgggc caaacaggat atctgtggta agcagttcct gccccggctc agggccaaga
      181 acagatggtc cccagatgcg gtcccgccct cagcagtttc tagagaacca tcagatgttt
      241 ccagggtgcc ccaaggacct gaaatgaccc tgtgccttat ttgaactaac caatcagttc
      301 gcttctcgct tctgttcgcg cgcttctgct ccccgagctc aataaaagag cccacaaccc
      361 ctcactcggc gcgccagtcc tccgatagac tgcgtcgccc gggtacccgt attcccaata
      421 aagcctcttg ctgtttgcat ccgaatcgtg gactcgctga tccttgggag ggtctcctca
      481 gattgattga ctgcccacct cgggggtctt tcatttggag gttccaccga gatttggaga
      541 cccctgccca gggaccaccg acccccccgc cgggaggtaa gctggccagc ggtcgtttcg
      601 tgtctgtctc tgtctttgtg cgtgtttgtg ccggcatcta atgtttgcgc ctgcgtctgt
      661 actagttagc taactagctc tgtatctggc ggacccgtgg tggaactgac gagttctgaa
      721 cacccggccg caaccctggg agacgtccca gggactttgg gggccgtttt tgtggcccga
      781 cctgaggaag ggagtcgatg tggaatccga ccccgtcagg atatgtggtt ctggtaggag
      841 acgagaacct aaaacagttc ccgcctccgt ctgaattttt gctttcggtt tggaaccgaa
      901 gccgcgcgtc ttgtctgctg cagcgctgca gcatcgttct gtgttgtctc tgtctgactg
      961 tgtttctgta tttgtctgaa aattagggcc agactgttac cactccctta agtttgacct
     1021 taggtcactg gaaagatgtc gagcggatcg ctcacaacca gtcggtagat gtcaagaaga
     1081 gacgttgggt taccttctgc tctgcagaat ggccaacctt taacgtcgga tggccgcgag
     1141 acggcacctt taaccgagac ctcatcaccc aggttaagat caaggtcttt tcacctggcc
     1201 cgcatggaca cccagaccag gtcccctaca tcgtgacctg ggaagccttg gcttttgacc
     1261 cccctccctg ggtcaagccc tttgtacacc ctaagcctcc gcctcctctt cctccatccg
     1321 ccccgtctct cccccttgaa cctcctcgtt cgaccccgcc tcgatcctcc ctttatccag
     1381 ccctcactcc ttctctaggc gccggaatta gatccccccg agtccaacac ccgtgggaat
     1441 cccatgggca ccatggcccc tcgctccaaa aatgctttcg cgtcgcgcag acactgctcg
     1501 gtagtttcgg ggatcagcgt ttgagtaaga gcccgcgtct gaaccctccg cgccgccccg
     1561 gccccagtgg aaagacgcgc aggcaaaacg caccacgtga cggagcgtga ccgcgcgccg
     1621 agcgcgcgcc agggtcgggc aggaagaggg cctatttccc atgattcctt catatttgca
     1681 tatacgatac aaggctgtta gagagataat tagaattaat ttgactgtaa acacaaagat
     1741 attagtacaa aatacgtgac gtagaaagta ataatttctt gggtagtttg cagttttaaa
     1801 attatgtttt aaaatggact atcatatgct taccgtaact tgaaagtatt tcgatttctt
     1861 ggctttatat atcttgtgga aaggacgaaa caccgtgctc gcttcggcag cacatatact
     1921 agtcgactag ggataacagg gtaattgttt gaatgaggct tcagtacttt acagaatcgt
     1981 tgcctgcaca tcttggaaac acttgctggg attacttctt caggttaacc caacagaagg
     2041 ctcgagcaac cagaattcaa ggggctactt taggagcaat tatcttgttt actaaaactg
     2101 aataccttgc tatctctttg atacattttt acaaagctga attaaaatgg tataaattaa
     2161 atcacttttt tcaattctac cgggtagggg aggcgctttt cccaaggcag tctggagcat
     2221 gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac
     2281 attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac
     2341 cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga
     2401 cgtgacaaat ggaagtagca cgtctcacta gtctcgtgca gatggacagc accgctgagc
     2461 aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct
     2521 gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg ctcaggggcg
     2581 gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt
     2641 ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctgcagc ccaagcttac
     2701 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc ccagggccgt
     2761 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgatccgga
     2821 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     2881 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     2941 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     3001 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     3061 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg
     3121 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     3181 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     3241 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgcctg
     3301 acgcccgccc cacgacccgc agcgcccgac cgaaaggagc gcacgacccc atgcatcgat
     3361 aaaataaaag attttattta gtctccagaa aaagggggga atgaaagacc ccacctgtag
     3421 gtttggcaag ctagcttaag taacgccatt ttgcaaggca tggaaaatac ataactgaga
     3481 atagagaagt tcagatcaag gttaggaaca gagagacagc agaatatggg ccaaacagga
     3541 tatctgtggt aagcagttcc tgccccggct cagggccaag aacagatggt ccccagatgc
     3601 ggtcccgccc tcagcagttt ctagagaacc atcagatgtt tccagggtgc cccaaggacc
     3661 tgaaatgacc ctgtgcctta tttgaactaa ccaatcagtt cgcttctcgc ttctgttcgc
     3721 gcgcttctgc tccccgagct caataaaaga gcccacaacc cctcactcgg cgcgccagtc
     3781 ctccgataga ctgcgtcgcc cgggtacccg tgtatccaat aaaccctctt gcagttgcat
     3841 ccgacttgtg gtctcgctgt tccttgggag ggtctcctct gagtgattga ctacccgtca
     3901 gcgggggtct ttcatgggta acagtttctt gaagttggag aacaacattc tgagggtagg
     3961 agtcgaatat taagtaatcc tgactcaatt agccactgtt ttgaatccac atactccaat
     4021 actcctgaaa tagttcatta tggacagcgc agaaagagct ggggagaatt gtgaaattgt
     4081 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     4141 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     4201 ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg
     4261 cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg
     4321 cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat
     4381 aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc
     4441 gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc
     4501 tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga
     4561 agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt
     4621 ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg
     4681 taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc
     4741 gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg
     4801 gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc
     4861 ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg
     4921 ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc
     4981 gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct
     5041 caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt
     5101 taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa
     5161 aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa
     5221 tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc
     5281 tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct
     5341 gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca
     5401 gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt
     5461 aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt
     5521 gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc
     5581 ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc
     5641 tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt
     5701 atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact
     5761 ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc
     5821 ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt
     5881 ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg
     5941 atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct
     6001 gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa
     6061 tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt
     6121 ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc
     6181 acatttcccc gaaaagtgcc acctgacgtc taagaaacca ttattatcat gacattaacc
     6241 tataaaaata ggcgtatcac gaggcccttt cgtctcgcgc gtttcggtga tgacggtgaa
     6301 aacctctgac acatgcagct cccggagacg gtcacagctt gtctgtaagc ggatgccggg
     6361 agcagacaag cccgtcaggg cgcgtcagcg ggtgttggcg ggtgtcgggg ctggcttaac
     6421 tatgcggcat cagagcagat tgtactgaga gtgcaccata tgcggtgtga aataccgcac
     6481 agatgcgtaa ggagaaaata ccgcatcagg cgccattcgc cattcaggct gcgcaactgt
     6541 tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt
     6601 gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg
     6661 acggcgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     6721 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     6781 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     6841 cggccacgat gcgtccggcg tagaggcgat tagtccaatt tgttaaagac aggatatcag
     6901 tggtccaggc tctagttttg actcaacaat atcaccagct gaagcctata gagtacgagc
     6961 catagataaa ataaaagatt ttatttagtc tccagaaaaa ggggggaa
//
