LOCUS       sequence-364598-        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-shMMERVK10c_1
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     133..152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     386..403
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    520..541
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(556..561,562..579,580..586)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    594..610
                     /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     599..621
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     618..634
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     618..634
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     653..673
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        655..673
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        701..927
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             928..1108
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1155..1280
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1773..2006
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2191..2235
                     /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        2533..2781
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2533..2553
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2704..2723
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2878..2995
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        3044..3554
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3473..3491
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3576..4175
                     /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(3576..3595)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4072..4092
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             4303..4536
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4608..4729
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4645..4664)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4699..4718
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4769..4904
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4924..4943)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4925..4943)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4953..4970)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4953..4969)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4962..4984)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5111..5566
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5198..5217)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5408..5429
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5592..5696
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5697..5765,5766..6557)
                     /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(5915..5934)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(6728..7084,1..232)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
       61 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
      121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
      181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
      241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
      301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccgc
      361 tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg aagagcgccc
      421 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
      481 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
      541 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
      601 cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc gcgcaattaa
      661 ccctcactaa agggaacaaa agctggagct gcaagcttaa tgtagtctta tgcaatactc
      721 ttgtagtctt gcaacatggt aacgatgagt tagcaacatg ccttacaagg agagaaaaag
      781 caccgtgcat gccgattggt ggaagtaagg tggtacgatc gtgccttatt aggaaggcaa
      841 cagacgggtc tgacatggat tggacgaacc actgaattgc cgcattgcag agatattgta
      901 tttaagtgcc tagctcgata cataaacggg tctctctggt tagaccagat ctgagcctgg
      961 gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg
     1021 cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc
     1081 ttttagtcag tgtggaaaat ctctagcagt ggcgcccgaa cagggacttg aaagcgaaag
     1141 ggaaaccaga ggagctctct cgacgcagga ctcggcttgc tgaagcgcgc acggcaagag
     1201 gcgaggggcg gcgactggtg agtacgccaa aaattttgac tagcggaggc tagaaggaga
     1261 gagatgggtg cgagagcgtc agtattaagc gggggagaat tagatcgcga tgggaaaaaa
     1321 ttcggttaag gccaggggga aagaaaaaat ataaattaaa acatatagta tgggcaagca
     1381 gggagctaga acgattcgca gttaatcctg gcctgttaga aacatcagaa ggctgtagac
     1441 aaatactggg acagctacaa ccatcccttc agacaggatc agaagaactt agatcattat
     1501 ataatacagt agcaaccctc tattgtgtgc atcaaaggat agagataaaa gacaccaagg
     1561 aagctttaga caagatagag gaagagcaaa acaaaagtaa gaccaccgca cagcaagcgg
     1621 ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag tgaattatat
     1681 aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc aaagagaaga
     1741 gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg gttcttggga
     1801 gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc cagacaatta
     1861 ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc gcaacagcat
     1921 ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct ggctgtggaa
     1981 agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa actcatttgc
     2041 accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca gatttggaat
     2101 cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt aatacactcc
     2161 ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt ggaattagat
     2221 aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta tataaaatta
     2281 ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt actttctata
     2341 gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct cccaaccccg
     2401 aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga cagagacaga
     2461 tccattcgat tagtgaacgg atctcgacgg tatcgatcac gagactagcc tcgagcggcc
     2521 gcccccttca ccgagggcct atttcccatg attccttcat atttgcatat acgatacaag
     2581 gctgttagag agataattgg aattaatttg actgtaaaca caaagatatt agtacaaaat
     2641 acgtgacgta gaaagtaata atttcttggg tagtttgcag ttttaaaatt atgttttaaa
     2701 atggactatc atatgcttac cgtaacttga aagtatttcg atttcttggc tttatatatc
     2761 ttgtggaaag gacgaaacac cggccggtaa gtgtaaagaa attactcgag taatttcttt
     2821 acacttaccg gtttttgaat tctcgacctc gagacaaatg gcagtattca tccacaattt
     2881 taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag acataatagc
     2941 aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa attttcgggt
     3001 ttattacagg gacagcagag atccactttg gccgcggctc gagggggttg gggttgcgcc
     3061 ttttccaagg cagccctggg tttgcgcagg gacgcggctg ctctgggcgt ggttccggga
     3121 aacgcagcgg cgccgaccct gggtctcgca cattcttcac gtccgttcgc agcgtcaccc
     3181 ggatcttcgc cgctaccctt gtgggccccc cggcgacgct tcctgctccg cccctaagtc
     3241 gggaaggttc cttgcggttc gcggcgtgcc ggacgtgaca aacggaagcc gcacgtctca
     3301 ctagtaccct cgcagacgga cagcgccagg gagcaatggc agcgcgccga ccgcgatggg
     3361 ctgtggccaa tagcggctgc tcagcagggc gcgccgagag cagcggccgg gaaggggcgg
     3421 tgcgggaggc ggggtgtggg gcggtagtgt gggccctgtt cctgcccgcg cggtgttccg
     3481 cattctgcaa gcctccggag cgcacgtcgg cagtcggctc cctcgttgac cgaatcaccg
     3541 acctctctcc ccagggggat ccaccggagc ttaccatgac cgagtacaag cccacggtgc
     3601 gcctcgccac ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg
     3661 actaccccgc cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc
     3721 tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg
     3781 acggcgccgc ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg
     3841 ccgagatcgg cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga
     3901 tggaaggcct cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg
     3961 gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg
     4021 aggcggccga gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc
     4081 ccttctacga gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc
     4141 gcacctggtg catgacccgc aagcccggtg cctgacgccc gccccacgac ccgcagcgcc
     4201 cgaccgaaag gagcgcacga ccccatgcat cggtaccttt aagaccaatg acttacaagg
     4261 cagctgtaga tcttagccac tttttaaaag aaaagggggg actggaaggg ctaattcact
     4321 cccaacgaag acaagatctg ctttttgctt gtactgggtc tctctggtta gaccagatct
     4381 gagcctggga gctctctggc taactaggga acccactgct taagcctcaa taaagcttgc
     4441 cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc
     4501 tcagaccctt ttagtcagtg tggaaaatct ctagcagtag tagttcatgt catcttatta
     4561 ttcagtattt ataacttgca aagaaatgaa tatcagagag tgagaggaac ttgtttattg
     4621 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     4681 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctggc
     4741 tctagctatc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     4801 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     4861 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggga cgtacccaat
     4921 tcgccctata gtgagtcgta ttacgcgcgc tcactggccg tcgttttaca acgtcgtgac
     4981 tgggaaaacc ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc
     5041 tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat
     5101 ggcgaatggg acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     5161 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     5221 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     5281 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     5341 cgtagtgggc catcgccctg atagacggtt tttcgccctt tgacgttgga gtccacgttc
     5401 tttaatagtg gactcttgtt ccaaactgga acaacactca accctatctc ggtctattct
     5461 tttgatttat aagggatttt gccgatttcg gcctattggt taaaaaatga gctgatttaa
     5521 caaaaattta acgcgaattt taacaaaata ttaacgctta caatttaggt ggcacttttc
     5581 ggggaaatgt gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc
     5641 cgctcatgag acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga
     5701 gtattcaaca tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt
     5761 ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag
     5821 tgggttacat cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag
     5881 aacgttttcc aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta
     5941 ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg
     6001 agtactcacc agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca
     6061 gtgctgccat aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag
     6121 gaccgaagga gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc
     6181 gttgggaacc ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg
     6241 tagcaatggc aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc
     6301 ggcaacaatt aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg
     6361 cccttccggc tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg
     6421 gtatcattgc agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga
     6481 cggggagtca ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac
     6541 tgattaagca ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa
     6601 aacttcattt ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca
     6661 aaatccctta acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag
     6721 gatcttcttg agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac
     6781 cgctaccagc ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa
     6841 ctggcttcag cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc
     6901 accacttcaa gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag
     6961 tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac
     7021 cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc
     7081 gaac
//
