LOCUS       sequence-177702-        7088 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To establish Dsup knock down cells.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7088)
  AUTHORS   Hashimoto T, Horikawa DD, Saito Y, Kuwahara H, Kozuka-Hata H, Shin-I
            T, Minakuchi Y, Ohishi K, Motoyama A, Aizu T, Enomoto A, Kondo K, 
            Tanaka S, Hara Y, Koshikawa S, Sagara H, Miura T, Yokobori S, 
            Miyagawa K, Suzuki Y, Kubo T, Oyama M, Kohara Y, Fujiyama A, Arakawa
            K, Katayama T, Toyoda A, Kunieda T
  TITLE     Extremotolerant tardigrade genome and improved radiotolerance of 
            human cultured cells by tardigrade-unique protein.
  JOURNAL   Nat Commun. 2016 Sep 20;7:12808. doi: 10.1038/ncomms12808.
  PUBMED    27649274
REFERENCE   2  (bases 1 to 7088)
  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"; journalName: "Nat 
            Commun."; date: "2016-09-20"; volume: "7:12808. doi"; pages: " 
            10.1038/ncomms12808"
COMMENT     Sequence Label: pLKO.1-shDsup
FEATURES             Location/Qualifiers
     source          1..7088
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     469..488
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     promoter        complement(510..1020)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(573..591)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     misc_feature    1069..1186
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        complement(1287..1535)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(1345..1364)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1515..1535)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     CDS             complement(1833..1877)
                     /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    2062..2295
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     misc_feature    2788..2913
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     LTR             2960..3140
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     promoter        complement(3141..3367)
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     primer_bind     complement(3395..3415)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3395..3413)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3434..3450)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3434..3450)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3447..3469)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3458..3474
                     /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(3482..3488,3489..3506,3507..3512))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3527..3548
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3665..3682)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3836..4424)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3916..3935)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4595..5386,5387..5455))
                     /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     5218..5237
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5456..5560)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5586..6041)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5723..5744)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5935..5954
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6168..6190
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6182..6199
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6183..6199
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     6209..6228
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6209..6227
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     rep_origin      6248..6383
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6423..6544
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6434..6453)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     6488..6507
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     LTR             6616..6849
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     CDS             complement(join(6977..7088,1..488))
                     /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(7060..7080)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
ORIGIN
        1 gaggtctcca ggaaggcggg caccccggcg cgctcggccg cctccactcc ggggagcacg
       61 acggcgctgc ccagaccctt gccctggtgg tcgggcgaga cgccgacggt ggccaggaac
      121 cacgcgggct ccttgggccg gtgcggcgcc aggaggcctt ccatctgttg ctgcgcggcc
      181 agccgggaac cgctcaactc ggccatgcgc gggccgatct cggcgaacac cgcccccgct
      241 tcgacgctct ccggcgtggt ccagaccgcc accgcggcgc cgtcgtccgc gacccacacc
      301 ttgccgatgt cgagcccgac gcgcgtgagg aagagttctt gcagctcggt gacccgctcg
      361 atgtggcggt ccggatcgac ggtgtggcgc gtggcggggt agtcggcgaa cgcggcggcg
      421 agggtgcgta cggccctggg gacgtcgtcg cgggtggcga ggcgcaccgt gggcttgtac
      481 tcggtcatgg taagctccgg tggatccccc tggggagaga ggtcggtgat tcggtcaacg
      541 agggagccga ctgccgacgt gcgctccgga ggcttgcaga atgcggaaca ccgcgcgggc
      601 aggaacaggg cccacactac cgccccacac cccgcctccc gcaccgcccc ttcccggccg
      661 ctgctctcgg cgcgccctgc tgagcagccg ctattggcca cagcccatcg cggtcggcgc
      721 gctgccattg ctccctggcg ctgtccgtct gcgagggtac tagtgagacg tgcggcttcc
      781 gtttgtcacg tccggcacgc cgcgaaccgc aaggaacctt cccgacttag gggcggagca
      841 ggaagcgtcg ccggggggcc cacaagggta gcggcgaaga tccgggtgac gctgcgaacg
      901 gacgtgaaga atgtgcgaga cccagggtcg gcgccgctgc gtttcccgga accacgccca
      961 gagcagccgc gtccctgcgc aaacccaggg ctgccttgga aaaggcgcaa ccccaacccc
     1021 ctcgagccgc ggccaaagtg gatctctgct gtccctgtaa taaacccgaa aattttgaat
     1081 ttttgtaatt tgtttttgta attctttagt ttgtatgtct gttgctatta tgtctactat
     1141 tctttcccct gcactgtacc ccccaatccc cccttttctt ttaaaattgt ggatgaatac
     1201 tgccatttgt ctcgaggtcg agaattcaaa aagaacgtaa ccgttaccaa aggtctcttg
     1261 aacctttggt aacggttacg ttcaccggtg tttcgtcctt tccacaagat atataaagcc
     1321 aagaaatcga aatactttca agttacggta agcatatgat agtccatttt aaaacataat
     1381 tttaaaactg caaactaccc aagaaattat tactttctac gtcacgtatt ttgtactaat
     1441 atctttgtgt ttacagtcaa attaattcca attatctctc taacagcctt gtatcgtata
     1501 tgcaaatatg aaggaatcat gggaaatagg ccctcggtga agggggcggc cgctcgaggc
     1561 tagtctcgtg atcgataccg tcgagatccg ttcactaatc gaatggatct gtctctgtct
     1621 ctctctccac cttcttcttc tattccttcg ggcctgtcgg gtcccctcgg ggttgggagg
     1681 tgggtctgaa acgataatgg tgaatatccc tgcctaactc tattcactat agaaagtaca
     1741 gcaaaaacta ttcttaaacc taccaagcct cctactatca ttatgaataa ttttatatac
     1801 cacagccaat ttgttatgtt aaaccaattc cacaaacttg cccatttatc taattccaat
     1861 aattcttgtt cattcttttc ttgctggttt tgcgattctt caattaagga gtgtattaag
     1921 cttgtgtaat tgttaatttc tctgtcccac tccatccagg tcgtgtgatt ccaaatctgt
     1981 tccagagatt tattactcca actagcattc caaggcacag cagtggtgca aatgagtttt
     2041 ccagagcaac cccaaatccc caggagctgt tgatccttta ggtatctttc cacagccagg
     2101 attcttgcct ggagctgctt gatgccccag actgtgagtt gcaacagatg ctgttgcgcc
     2161 tcaatagccc tcagcaaatt gttctgctgc tgcactatac cagacaataa ttgtctggcc
     2221 tgtaccgtca gcgtcattga cgctgcgccc atagtgcttc ctgctgctcc caagaaccca
     2281 aggaacaaag ctcctattcc cactgctctt ttttctctct gcaccactct tctctttgcc
     2341 ttggtgggtg ctactcctaa tggttcaatt tttactactt tatatttata taattcactt
     2401 ctccaattgt ccctcatatc tcctcctcca ggtctgaaga tcagcggccg cttgctgtgc
     2461 ggtggtctta cttttgtttt gctcttcctc tatcttgtct aaagcttcct tggtgtcttt
     2521 tatctctatc ctttgatgca cacaatagag ggttgctact gtattatata atgatctaag
     2581 ttcttctgat cctgtctgaa gggatggttg tagctgtccc agtatttgtc tacagccttc
     2641 tgatgtttct aacaggccag gattaactgc gaatcgttct agctccctgc ttgcccatac
     2701 tatatgtttt aatttatatt ttttctttcc ccctggcctt aaccgaattt tttcccatcg
     2761 cgatctaatt ctcccccgct taatactgac gctctcgcac ccatctctct ccttctagcc
     2821 tccgctagtc aaaatttttg gcgtactcac cagtcgccgc ccctcgcctc ttgccgtgcg
     2881 cgcttcagca agccgagtcc tgcgtcgaga gagctcctct ggtttccctt tcgctttcaa
     2941 gtccctgttc gggcgccact gctagagatt ttccacactg actaaaaggg tctgagggat
     3001 ctctagttac cagagtcaca caacagacgg gcacacacta cttgaagcac tcaaggcaag
     3061 ctttattgag gcttaagcag tgggttccct agttagccag agagctccca ggctcagatc
     3121 tggtctaacc agagagaccc gtttatgtat cgagctaggc acttaaatac aatatctctg
     3181 caatgcggca attcagtggt tcgtccaatc catgtcagac ccgtctgttg ccttcctaat
     3241 aaggcacgat cgtaccacct tacttccacc aatcggcatg cacggtgctt tttctctcct
     3301 tgtaaggcat gttgctaact catcgttacc atgttgcaag actacaagag tattgcataa
     3361 gactacatta agcttgcagc tccagctttt gttcccttta gtgagggtta attgcgcgct
     3421 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     3481 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     3541 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     3601 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     3661 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     3721 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     3781 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     3841 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     3901 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     3961 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4021 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4081 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     4141 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     4201 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     4261 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     4321 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     4381 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     4441 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     4501 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     4561 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     4621 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     4681 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     4741 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     4801 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     4861 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     4921 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     4981 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5041 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     5101 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     5161 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     5221 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     5281 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     5341 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     5401 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     5461 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     5521 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     5581 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     5641 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     5701 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     5761 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     5821 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     5881 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     5941 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     6001 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
     6061 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     6121 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     6181 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac
     6241 gtccctaggc ctccaaaaaa gcctcctcac tacttctgga atagctcaga ggccgaggcg
     6301 gcctcggcct ctgcataaat aaaaaaaatt agtcagccat ggggcggaga atgggcggaa
     6361 ctgggcggag ttaggggcgg gatgggcgga gttaggggcg ggatagctag agccagacat
     6421 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     6481 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     6541 agttcctctc actctctgat attcatttct ttgcaagtta taaatactga ataataagat
     6601 gacatgaact actactgcta gagattttcc acactgacta aaagggtctg agggatctct
     6661 agttaccaga gtcacacaac agacgggcac acactacttg aagcactcaa ggcaagcttt
     6721 attgaggctt aagcagtggg ttccctagtt agccagagag ctcccaggct cagatctggt
     6781 ctaaccagag agacccagta caagcaaaaa gcagatcttg tcttcgttgg gagtgaatta
     6841 gcccttccag tccccccttt tcttttaaaa agtggctaag atctacagct gccttgtaag
     6901 tcattggtct taaaggtacc gatgcatggg gtcgtgcgct cctttcggtc gggcgctgcg
     6961 ggtcgtgggg cgggcgtcag gcaccgggct tgcgggtcat gcaccaggtg cgcggtcctt
     7021 cgggcacctc gacgtcggcg gtgacggtga agccgagccg ctcgtagaag gggaggttgc
     7081 ggggcgcg
//
