LOCUS       sequence-364610-        7084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  shRNA mediated knockdown.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7084)
  AUTHORS   Asimi V, Sampath Kumar A, Niskanen H, Riemenschneider C, Hetzel S, 
            Naderi J, Fasching N, Popitsch N, Du M, Kretzmer H, Smith ZD, 
            Weigert R, Walther M, Mamde S, Meierhofer D, Wittler L, Buschow R, 
            Timmermann B, Cisse II, Ameres SL, Meissner A, Hnisz D
  TITLE     Hijacking of transcriptional condensates by endogenous retroviruses.
  JOURNAL   Nat Genet. 2022 Aug;54(8):1238-1247. doi: 
            10.1038/s41588-022-01132-w. Epub 2022 Jul 21.
  PUBMED    35864192
REFERENCE   2  (bases 1 to 7084)
  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/s41588-022-01132-w"; journalName: "Nat Genet"; date: 
            "2022-08"; volume: "54"; issue: "8"; pages: "1238-1247"
COMMENT     Sequence Label: pLKO.1-Tet-Puro-shMMERVK9c_1
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     132..151
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     385..402
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    519..540
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(555..560,561..578,579..585)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    593..609
                     /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)."
     primer_bind     598..620
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     617..633
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     617..633
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     652..672
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        654..672
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        700..926
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             927..1107
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1154..1279
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1772..2005
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2190..2234
                     /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"
     promoter        2532..2780
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2532..2552
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2703..2722
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2877..2994
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3043..3553
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3472..3490
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3575..4174
                     /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(3575..3594)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4071..4091
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4302..4535
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4607..4728
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4644..4663)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4698..4717
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4768..4903
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4923..4942)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4924..4942)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4952..4969)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4952..4968)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4961..4983)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5110..5565
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5197..5216)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5407..5428
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5591..5695
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5696..5764,5765..6556)
                     /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(5914..5933)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6727..7084,1..231)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
       61 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
      121 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
      181 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
      241 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
      301 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
      361 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgccca
      421 atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg
      481 tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat
      541 taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc
      601 ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagcg cgcaattaac
      661 cctcactaaa gggaacaaaa gctggagctg caagcttaat gtagtcttat gcaatactct
      721 tgtagtcttg caacatggta acgatgagtt agcaacatgc cttacaagga gagaaaaagc
      781 accgtgcatg ccgattggtg gaagtaaggt ggtacgatcg tgccttatta ggaaggcaac
      841 agacgggtct gacatggatt ggacgaacca ctgaattgcc gcattgcaga gatattgtat
      901 ttaagtgcct agctcgatac ataaacgggt ctctctggtt agaccagatc tgagcctggg
      961 agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg ccttgagtgc
     1021 ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc ctcagaccct
     1081 tttagtcagt gtggaaaatc tctagcagtg gcgcccgaac agggacttga aagcgaaagg
     1141 gaaaccagag gagctctctc gacgcaggac tcggcttgct gaagcgcgca cggcaagagg
     1201 cgaggggcgg cgactggtga gtacgccaaa aattttgact agcggaggct agaaggagag
     1261 agatgggtgc gagagcgtca gtattaagcg ggggagaatt agatcgcgat gggaaaaaat
     1321 tcggttaagg ccagggggaa agaaaaaata taaattaaaa catatagtat gggcaagcag
     1381 ggagctagaa cgattcgcag ttaatcctgg cctgttagaa acatcagaag gctgtagaca
     1441 aatactggga cagctacaac catcccttca gacaggatca gaagaactta gatcattata
     1501 taatacagta gcaaccctct attgtgtgca tcaaaggata gagataaaag acaccaagga
     1561 agctttagac aagatagagg aagagcaaaa caaaagtaag accaccgcac agcaagcggc
     1621 cgctgatctt cagacctgga ggaggagata tgagggacaa ttggagaagt gaattatata
     1681 aatataaagt agtaaaaatt gaaccattag gagtagcacc caccaaggca aagagaagag
     1741 tggtgcagag agaaaaaaga gcagtgggaa taggagcttt gttccttggg ttcttgggag
     1801 cagcaggaag cactatgggc gcagcgtcaa tgacgctgac ggtacaggcc agacaattat
     1861 tgtctggtat agtgcagcag cagaacaatt tgctgagggc tattgaggcg caacagcatc
     1921 tgttgcaact cacagtctgg ggcatcaagc agctccaggc aagaatcctg gctgtggaaa
     1981 gatacctaaa ggatcaacag ctcctgggga tttggggttg ctctggaaaa ctcatttgca
     2041 ccactgctgt gccttggaat gctagttgga gtaataaatc tctggaacag atttggaatc
     2101 acacgacctg gatggagtgg gacagagaaa ttaacaatta cacaagctta atacactcct
     2161 taattgaaga atcgcaaaac cagcaagaaa agaatgaaca agaattattg gaattagata
     2221 aatgggcaag tttgtggaat tggtttaaca taacaaattg gctgtggtat ataaaattat
     2281 tcataatgat agtaggaggc ttggtaggtt taagaatagt ttttgctgta ctttctatag
     2341 tgaatagagt taggcaggga tattcaccat tatcgtttca gacccacctc ccaaccccga
     2401 ggggacccga caggcccgaa ggaatagaag aagaaggtgg agagagagac agagacagat
     2461 ccattcgatt agtgaacgga tctcgacggt atcgatcacg agactagcct cgagcggccg
     2521 cccccttcac cgagggccta tttcccatga ttccttcata tttgcatata cgatacaagg
     2581 ctgttagaga gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata
     2641 cgtgacgtag aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa
     2701 tggactatca tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct
     2761 tgtggaaagg acgaaacacc ggcccagagg cagagagaaa tttctcgaga aatttctctc
     2821 tgcctctggg tttttgaatt ctcgacctcg agacaaatgg cagtattcat ccacaatttt
     2881 aaaagaaaag gggggattgg ggggtacagt gcaggggaaa gaatagtaga cataatagca
     2941 acagacatac aaactaaaga attacaaaaa caaattacaa aaattcaaaa ttttcgggtt
     3001 tattacaggg acagcagaga tccactttgg ccgcggctcg agggggttgg ggttgcgcct
     3061 tttccaaggc agccctgggt ttgcgcaggg acgcggctgc tctgggcgtg gttccgggaa
     3121 acgcagcggc gccgaccctg ggtctcgcac attcttcacg tccgttcgca gcgtcacccg
     3181 gatcttcgcc gctacccttg tgggcccccc ggcgacgctt cctgctccgc ccctaagtcg
     3241 ggaaggttcc ttgcggttcg cggcgtgccg gacgtgacaa acggaagccg cacgtctcac
     3301 tagtaccctc gcagacggac agcgccaggg agcaatggca gcgcgccgac cgcgatgggc
     3361 tgtggccaat agcggctgct cagcagggcg cgccgagagc agcggccggg aaggggcggt
     3421 gcgggaggcg gggtgtgggg cggtagtgtg ggccctgttc ctgcccgcgc ggtgttccgc
     3481 attctgcaag cctccggagc gcacgtcggc agtcggctcc ctcgttgacc gaatcaccga
     3541 cctctctccc cagggggatc caccggagct taccatgacc gagtacaagc ccacggtgcg
     3601 cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg cgttcgccga
     3661 ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg tcaccgagct
     3721 gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg tcgcggacga
     3781 cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg cggtgttcgc
     3841 cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc agcaacagat
     3901 ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg ccaccgtcgg
     3961 cgtctcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc ccggagtgga
     4021 ggcggccgag cgcgccgggg tgcccgcctt cctggagacc tccgcgcccc gcaacctccc
     4081 cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg aaggaccgcg
     4141 cacctggtgc atgacccgca agcccggtgc ctgacgcccg ccccacgacc cgcagcgccc
     4201 gaccgaaagg agcgcacgac cccatgcatc ggtaccttta agaccaatga cttacaaggc
     4261 agctgtagat cttagccact ttttaaaaga aaagggggga ctggaagggc taattcactc
     4321 ccaacgaaga caagatctgc tttttgcttg tactgggtct ctctggttag accagatctg
     4381 agcctgggag ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc
     4441 ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct
     4501 cagacccttt tagtcagtgt ggaaaatctc tagcagtagt agttcatgtc atcttattat
     4561 tcagtattta taacttgcaa agaaatgaat atcagagagt gagaggaact tgtttattgc
     4621 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     4681 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggct
     4741 ctagctatcc cgcccctaac tccgcccatc ccgcccctaa ctccgcccag ttccgcccat
     4801 tctccgcccc atggctgact aatttttttt atttatgcag aggccgaggc cgcctcggcc
     4861 tctgagctat tccagaagta gtgaggaggc ttttttggag gcctagggac gtacccaatt
     4921 cgccctatag tgagtcgtat tacgcgcgct cactggccgt cgttttacaa cgtcgtgact
     4981 gggaaaaccc tggcgttacc caacttaatc gccttgcagc acatccccct ttcgccagct
     5041 ggcgtaatag cgaagaggcc cgcaccgatc gcccttccca acagttgcgc agcctgaatg
     5101 gcgaatggga cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     5161 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     5221 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     5281 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     5341 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct
     5401 ttaatagtgg actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt
     5461 ttgatttata agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac
     5521 aaaaatttaa cgcgaatttt aacaaaatat taacgcttac aatttaggtg gcacttttcg
     5581 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     5641 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     5701 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     5761 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     5821 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     5881 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     5941 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     6001 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     6061 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     6121 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     6181 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     6241 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     6301 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     6361 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     6421 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     6481 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     6541 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     6601 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     6661 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     6721 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     6781 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     6841 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     6901 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6961 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     7021 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     7081 aacg
//
