LOCUS       sequence-364612-        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_2
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(13..18,19..36,37..43)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    51..67
                     /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     56..78
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     75..91
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     75..91
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     110..130
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        112..130
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        158..384
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             385..565
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    612..737
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1230..1463
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1648..1692
                     /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        1990..2238
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1990..2010
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2161..2180
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2335..2452
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2501..3011
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     2930..2948
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3033..3632
                     /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(3033..3052)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3529..3549
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3760..3993
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4065..4186
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4102..4121)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4156..4175
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4226..4361
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4381..4400)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4382..4400)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4410..4427)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4410..4426)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4419..4441)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4568..5023
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4655..4674)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4865..4886
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5049..5153
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5154..5222,5223..6014)
                     /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(5372..5391)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6185..6773
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6674..6693
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6927..6944
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    7061..7082
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg
       61 ataacaattt cacacaggaa acagctatga ccatgattac gccaagcgcg caattaaccc
      121 tcactaaagg gaacaaaagc tggagctgca agcttaatgt agtcttatgc aatactcttg
      181 tagtcttgca acatggtaac gatgagttag caacatgcct tacaaggaga gaaaaagcac
      241 cgtgcatgcc gattggtgga agtaaggtgg tacgatcgtg ccttattagg aaggcaacag
      301 acgggtctga catggattgg acgaaccact gaattgccgc attgcagaga tattgtattt
      361 aagtgcctag ctcgatacat aaacgggtct ctctggttag accagatctg agcctgggag
      421 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
      481 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
      541 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
      601 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
      661 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
      721 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
      781 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
      841 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
      901 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
      961 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     1021 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
     1081 ctgatcttca gacctggagg aggagatatg agggacaatt ggagaagtga attatataaa
     1141 tataaagtag taaaaattga accattagga gtagcaccca ccaaggcaaa gagaagagtg
     1201 gtgcagagag aaaaaagagc agtgggaata ggagctttgt tccttgggtt cttgggagca
     1261 gcaggaagca ctatgggcgc agcgtcaatg acgctgacgg tacaggccag acaattattg
     1321 tctggtatag tgcagcagca gaacaatttg ctgagggcta ttgaggcgca acagcatctg
     1381 ttgcaactca cagtctgggg catcaagcag ctccaggcaa gaatcctggc tgtggaaaga
     1441 tacctaaagg atcaacagct cctggggatt tggggttgct ctggaaaact catttgcacc
     1501 actgctgtgc cttggaatgc tagttggagt aataaatctc tggaacagat ttggaatcac
     1561 acgacctgga tggagtggga cagagaaatt aacaattaca caagcttaat acactcctta
     1621 attgaagaat cgcaaaacca gcaagaaaag aatgaacaag aattattgga attagataaa
     1681 tgggcaagtt tgtggaattg gtttaacata acaaattggc tgtggtatat aaaattattc
     1741 ataatgatag taggaggctt ggtaggttta agaatagttt ttgctgtact ttctatagtg
     1801 aatagagtta ggcagggata ttcaccatta tcgtttcaga cccacctccc aaccccgagg
     1861 ggacccgaca ggcccgaagg aatagaagaa gaaggtggag agagagacag agacagatcc
     1921 attcgattag tgaacggatc tcgacggtat cgatcacgag actagcctcg agcggccgcc
     1981 cccttcaccg agggcctatt tcccatgatt ccttcatatt tgcatatacg atacaaggct
     2041 gttagagaga taattggaat taatttgact gtaaacacaa agatattagt acaaaatacg
     2101 tgacgtagaa agtaataatt tcttgggtag tttgcagttt taaaattatg ttttaaaatg
     2161 gactatcata tgcttaccgt aacttgaaag tatttcgatt tcttggcttt atatatcttg
     2221 tggaaaggac gaaacaccgg ttctctgcaa tcgtgattat tctcgagaat aatcacgatt
     2281 gcagagaatt tttgaattct cgacctcgag acaaatggca gtattcatcc acaattttaa
     2341 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     2401 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     2461 ttacagggac agcagagatc cactttggcc gcggctcgag ggggttgggg ttgcgccttt
     2521 tccaaggcag ccctgggttt gcgcagggac gcggctgctc tgggcgtggt tccgggaaac
     2581 gcagcggcgc cgaccctggg tctcgcacat tcttcacgtc cgttcgcagc gtcacccgga
     2641 tcttcgccgc tacccttgtg ggccccccgg cgacgcttcc tgctccgccc ctaagtcggg
     2701 aaggttcctt gcggttcgcg gcgtgccgga cgtgacaaac ggaagccgca cgtctcacta
     2761 gtaccctcgc agacggacag cgccagggag caatggcagc gcgccgaccg cgatgggctg
     2821 tggccaatag cggctgctca gcagggcgcg ccgagagcag cggccgggaa ggggcggtgc
     2881 gggaggcggg gtgtggggcg gtagtgtggg ccctgttcct gcccgcgcgg tgttccgcat
     2941 tctgcaagcc tccggagcgc acgtcggcag tcggctccct cgttgaccga atcaccgacc
     3001 tctctcccca gggggatcca ccggagctta ccatgaccga gtacaagccc acggtgcgcc
     3061 tcgccacccg cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact
     3121 accccgccac gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc
     3181 aagaactctt cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg
     3241 gcgccgcggt ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg
     3301 agatcggccc gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg
     3361 aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggcg
     3421 tctcgcccga ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg
     3481 cggccgagcg cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct
     3541 tctacgagcg gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca
     3601 cctggtgcat gacccgcaag cccggtgcct gacgcccgcc ccacgacccg cagcgcccga
     3661 ccgaaaggag cgcacgaccc catgcatcgg tacctttaag accaatgact tacaaggcag
     3721 ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta attcactccc
     3781 aacgaagaca agatctgctt tttgcttgta ctgggtctct ctggttagac cagatctgag
     3841 cctgggagct ctctggctaa ctagggaacc cactgcttaa gcctcaataa agcttgcctt
     3901 gagtgcttca agtagtgtgt gcccgtctgt tgtgtgactc tggtaactag agatccctca
     3961 gaccctttta gtcagtgtgg aaaatctcta gcagtagtag ttcatgtcat cttattattc
     4021 agtatttata acttgcaaag aaatgaatat cagagagtga gaggaacttg tttattgcag
     4081 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4141 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggctct
     4201 agctatcccg cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc
     4261 tccgccccat ggctgactaa ttttttttat ttatgcagag gccgaggccg cctcggcctc
     4321 tgagctattc cagaagtagt gaggaggctt ttttggaggc ctagggacgt acccaattcg
     4381 ccctatagtg agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg
     4441 gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg
     4501 cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc
     4561 gaatgggacg cgccctgtag cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc
     4621 gtgaccgcta cacttgccag cgccctagcg cccgctcctt tcgctttctt cccttccttt
     4681 ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc gggggctccc tttagggttc
     4741 cgatttagtg ctttacggca cctcgacccc aaaaaacttg attagggtga tggttcacgt
     4801 agtgggccat cgccctgata gacggttttt cgccctttga cgttggagtc cacgttcttt
     4861 aatagtggac tcttgttcca aactggaaca acactcaacc ctatctcggt ctattctttt
     4921 gatttataag ggattttgcc gatttcggcc tattggttaa aaaatgagct gatttaacaa
     4981 aaatttaacg cgaattttaa caaaatatta acgcttacaa tttaggtggc acttttcggg
     5041 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     5101 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     5161 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     5221 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     5281 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     5341 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     5401 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     5461 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     5521 ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg atcggaggac
     5581 cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc cttgatcgtt
     5641 gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg atgcctgtag
     5701 caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta gcttcccggc
     5761 aacaattaat agactggatg gaggcggata aagttgcagg accacttctg cgctcggccc
     5821 ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg tctcgcggta
     5881 tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc tacacgacgg
     5941 ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt gcctcactga
     6001 ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt gatttaaaac
     6061 ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa
     6121 tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat
     6181 cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc
     6241 taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg
     6301 gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc
     6361 acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg
     6421 ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg
     6481 ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa
     6541 cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg
     6601 aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga
     6661 gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct
     6721 gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca
     6781 gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttctttc
     6841 ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg
     6901 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
     6961 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     7021 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
     7081 atta
//
