LOCUS       sequence-364597-        7084 bp    DNA     circular SYN 15-APR-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 Apr 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-shMMETn_2
FEATURES             Location/Qualifiers
     source          1..7084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..44
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    161..182
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(197..202,203..220,221..227)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    235..251
                     /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     240..262
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     259..275
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     259..275
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     294..314
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        296..314
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        342..568
                     /label=RSV promoter
                     /note="Rous sarcoma virus enhancer/promoter"
     LTR             569..749
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    796..921
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1414..1647
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             1832..1876
                     /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        2174..2422
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     2174..2194
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2345..2364
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_feature    2519..2636
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2685..3195
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     3114..3132
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     CDS             3217..3816
                     /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(3217..3236)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3713..3733
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     LTR             3944..4177
                     /label=3' LTR (Delta-U3)
                     /note="self-inactivating 3' long terminal repeat (LTR) from
                     HIV-1"
     polyA_signal    4249..4370
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4286..4305)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4340..4359
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      4410..4545
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(4565..4584)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4566..4584)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4594..4611)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4594..4610)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4603..4625)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      4752..5207
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4839..4858)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5049..5070
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        5233..5337
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(5338..5406,5407..6198)
                     /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(5556..5575)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6369..6957
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6858..6877
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
       61 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
      121 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc
      181 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
      241 gcggataaca atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta
      301 accctcacta aagggaacaa aagctggagc tgcaagctta atgtagtctt atgcaatact
      361 cttgtagtct tgcaacatgg taacgatgag ttagcaacat gccttacaag gagagaaaaa
      421 gcaccgtgca tgccgattgg tggaagtaag gtggtacgat cgtgccttat taggaaggca
      481 acagacgggt ctgacatgga ttggacgaac cactgaattg ccgcattgca gagatattgt
      541 atttaagtgc ctagctcgat acataaacgg gtctctctgg ttagaccaga tctgagcctg
      601 ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt
      661 gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc
      721 cttttagtca gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa
      781 gggaaaccag aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga
      841 ggcgaggggc ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag
      901 agagatgggt gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa
      961 attcggttaa ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc
     1021 agggagctag aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga
     1081 caaatactgg gacagctaca accatccctt cagacaggat cagaagaact tagatcatta
     1141 tataatacag tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag
     1201 gaagctttag acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg
     1261 gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata
     1321 taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag
     1381 agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg
     1441 agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt
     1501 attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca
     1561 tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga
     1621 aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg
     1681 caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa
     1741 tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct taatacactc
     1801 cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga
     1861 taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt
     1921 attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat
     1981 agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc
     2041 gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag
     2101 atccattcga ttagtgaacg gatctcgacg gtatcgatca cgagactagc ctcgagcggc
     2161 cgcccccttc accgagggcc tatttcccat gattccttca tatttgcata tacgatacaa
     2221 ggctgttaga gagataattg gaattaattt gactgtaaac acaaagatat tagtacaaaa
     2281 tacgtgacgt agaaagtaat aatttcttgg gtagtttgca gttttaaaat tatgttttaa
     2341 aatggactat catatgctta ccgtaacttg aaagtatttc gatttcttgg ctttatatat
     2401 cttgtggaaa ggacgaaaca ccgggacatc caaacttcgt aatatctcga gatattacga
     2461 agtttggatg tctttttgaa ttctcgacct cgagacaaat ggcagtattc atccacaatt
     2521 ttaaaagaaa aggggggatt ggggggtaca gtgcagggga aagaatagta gacataatag
     2581 caacagacat acaaactaaa gaattacaaa aacaaattac aaaaattcaa aattttcggg
     2641 tttattacag ggacagcaga gatccacttt ggccgcggct cgagggggtt ggggttgcgc
     2701 cttttccaag gcagccctgg gtttgcgcag ggacgcggct gctctgggcg tggttccggg
     2761 aaacgcagcg gcgccgaccc tgggtctcgc acattcttca cgtccgttcg cagcgtcacc
     2821 cggatcttcg ccgctaccct tgtgggcccc ccggcgacgc ttcctgctcc gcccctaagt
     2881 cgggaaggtt ccttgcggtt cgcggcgtgc cggacgtgac aaacggaagc cgcacgtctc
     2941 actagtaccc tcgcagacgg acagcgccag ggagcaatgg cagcgcgccg accgcgatgg
     3001 gctgtggcca atagcggctg ctcagcaggg cgcgccgaga gcagcggccg ggaaggggcg
     3061 gtgcgggagg cggggtgtgg ggcggtagtg tgggccctgt tcctgcccgc gcggtgttcc
     3121 gcattctgca agcctccgga gcgcacgtcg gcagtcggct ccctcgttga ccgaatcacc
     3181 gacctctctc cccaggggga tccaccggag cttaccatga ccgagtacaa gcccacggtg
     3241 cgcctcgcca cccgcgacga cgtccccagg gccgtacgca ccctcgccgc cgcgttcgcc
     3301 gactaccccg ccacgcgcca caccgtcgat ccggaccgcc acatcgagcg ggtcaccgag
     3361 ctgcaagaac tcttcctcac gcgcgtcggg ctcgacatcg gcaaggtgtg ggtcgcggac
     3421 gacggcgccg cggtggcggt ctggaccacg ccggagagcg tcgaagcggg ggcggtgttc
     3481 gccgagatcg gcccgcgcat ggccgagttg agcggttccc ggctggccgc gcagcaacag
     3541 atggaaggcc tcctggcgcc gcaccggccc aaggagcccg cgtggttcct ggccaccgtc
     3601 ggcgtctcgc ccgaccacca gggcaagggt ctgggcagcg ccgtcgtgct ccccggagtg
     3661 gaggcggccg agcgcgccgg ggtgcccgcc ttcctggaga cctccgcgcc ccgcaacctc
     3721 cccttctacg agcggctcgg cttcaccgtc accgccgacg tcgaggtgcc cgaaggaccg
     3781 cgcacctggt gcatgacccg caagcccggt gcctgacgcc cgccccacga cccgcagcgc
     3841 ccgaccgaaa ggagcgcacg accccatgca tcggtacctt taagaccaat gacttacaag
     3901 gcagctgtag atcttagcca ctttttaaaa gaaaaggggg gactggaagg gctaattcac
     3961 tcccaacgaa gacaagatct gctttttgct tgtactgggt ctctctggtt agaccagatc
     4021 tgagcctggg agctctctgg ctaactaggg aacccactgc ttaagcctca ataaagcttg
     4081 ccttgagtgc ttcaagtagt gtgtgcccgt ctgttgtgtg actctggtaa ctagagatcc
     4141 ctcagaccct tttagtcagt gtggaaaatc tctagcagta gtagttcatg tcatcttatt
     4201 attcagtatt tataacttgc aaagaaatga atatcagaga gtgagaggaa cttgtttatt
     4261 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     4321 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgg
     4381 ctctagctat cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     4441 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     4501 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggg acgtacccaa
     4561 ttcgccctat agtgagtcgt attacgcgcg ctcactggcc gtcgttttac aacgtcgtga
     4621 ctgggaaaac cctggcgtta cccaacttaa tcgccttgca gcacatcccc ctttcgccag
     4681 ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc caacagttgc gcagcctgaa
     4741 tggcgaatgg gacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     4801 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     4861 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     4921 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     4981 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     5041 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     5101 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     5161 acaaaaattt aacgcgaatt ttaacaaaat attaacgctt acaatttagg tggcactttt
     5221 cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat
     5281 ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg
     5341 agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt
     5401 tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga
     5461 gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa
     5521 gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt
     5581 attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt
     5641 gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc
     5701 agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga
     5761 ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat
     5821 cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct
     5881 gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc
     5941 cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg
     6001 gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc
     6061 ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg
     6121 acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca
     6181 ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta gattgattta
     6241 aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc
     6301 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     6361 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     6421 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     6481 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
     6541 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     6601 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     6661 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     6721 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     6781 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     6841 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     6901 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     6961 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     7021 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     7081 accg
//
