LOCUS       sequence-269545-        7953 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of shRNAs, compatible with double-shRNA 
            expression, SFFV promoter, mCherry.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7953)
  AUTHORS   Kampmann M, Horlbeck MA, Chen Y, Tsai JC, Bassik MC, Gilbert LA, 
            Villalta JE, Kwon SC, Chang H, Kim VN, Weissman JS
  TITLE     Next-generation libraries for robust RNA interference-based 
            genome-wide screens.
  JOURNAL   Proc Natl Acad Sci U S A. 2015 Jun 30;112(26):E3384-91. doi: 
            10.1073/pnas.1508821112. Epub 2015 Jun 15.
  PUBMED    26080438
REFERENCE   2  (bases 1 to 7953)
  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: 
            "10.1073/pnas.1508821112"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2015-06-30- 30"; volume: "112"; issue: "26"; pages: 
            "E3384-91"
COMMENT     Sequence Label: pMK1221
FEATURES             Location/Qualifiers
     source          1..7953
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(67..86)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        258..637
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        639..837
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     792..812
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             855..1035
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1082..1207
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1704..1937
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2122..2166
                     /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    2464..2581
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2766..3173
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     CDS             3185..3781
                     /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(3185..3204)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3681..3701
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3791..3844
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3857..4567
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(4002..4020)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4270..4289
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     ncRNA           4659..4673
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     misc_feature    4862..5450
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4915..4935)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5333..5344)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5453..5469)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5454..5470)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5979..6159
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6221..6809)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6301..6320)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6980..7771,7772..7840))
                     /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     7603..7622
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7841..7945)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc tatggtgcac
       61 tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc ctgcttgtgt
      121 gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc aaggcttgac
      181 cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg
      241 ggccagatat acgcgttgac attgattatt gactagttat taatagtaat caattacggg
      301 gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg taaatggccc
      361 gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt atgttcccat
      421 agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac ggtaaactgc
      481 ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg acgtcaatga
      541 cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact ttcctacttg
      601 gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt ggcagtacat
      661 caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc ccattgacgt
      721 caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc gtaacaactc
      781 cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata taagcagcgc
      841 gttttgcctg tactgggtct ctctggttag accagatctg agcctgggag ctctctggct
      901 aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt
      961 gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt tagtcagtgt
     1021 ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga aaccagagga
     1081 gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg
     1141 actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag atgggtgcga
     1201 gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc
     1261 agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg agctagaacg
     1321 attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa tactgggaca
     1381 gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata atacagtagc
     1441 aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag ctttagacaa
     1501 gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg gccgctgatc
     1561 ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata taaatataaa
     1621 gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag agtggtgcag
     1681 agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg agcagcagga
     1741 agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt attgtctggt
     1801 atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca tctgttgcaa
     1861 ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga aagataccta
     1921 aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg caccactgct
     1981 gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa tcacacgacc
     2041 tggatggagt gggacagaga aattaacaat tacacaagct taatacactc cttaattgaa
     2101 gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga taaatgggca
     2161 agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt attcataatg
     2221 atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat agtgaataga
     2281 gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc gaggggaccc
     2341 gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag atccattcga
     2401 ttagtgaacg gatcggcact gcgtgcgcca attctgcaga caaatggcag tattcatcca
     2461 caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa tagtagacat
     2521 aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt
     2581 tcgggtttat tacagggaca gcagagatcc agtttggtta gtaccgggcc cgctctaggg
     2641 ccgcgtggat aaccgtatta ccgccatgca ttcctgcagc cccgataaaa taaaagattt
     2701 tatttagtct ccagaaaaag gggggaatga aagaccccac ctgtaggttt ggcaagctag
     2761 ctgcagtaac gccattttgc aaggcatgga aaaataccaa accaagaata gagaagttca
     2821 gatcaagggc gggtacatga aaatagctaa cgttgggcca aacaggatat ctgcggtgag
     2881 cagtttcggc cccggcccgg ggccaagaac agatggtcac cgcagtttcg gccccggccc
     2941 gaggccaaga acagatggtc cccagatatg gcccaaccct cagcagtttc ttaagaccca
     3001 tcagatgttt ccaggctccc ccaaggacct gaaatgaccc tgcgccttat ttgaattaac
     3061 caatcagcct gcttctcgct tctgttcgcg cgcttctgct tcccgagctc tataaaagag
     3121 ctcacaaccc ctcactcggc gcgccagtcc tccgacagac tgagtcgccc gggtctagcc
     3181 caccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tcccccgggc
     3241 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgaccc
     3301 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     3361 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     3421 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     3481 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     3541 ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct
     3601 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     3661 cctggagacc tccgcgcccc gcaacctccc cttctacgag cggctcggct tcaccgtcac
     3721 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     3781 cggatcggga gagggcagag gaagtctgct aacatgcggt gacgtcgagg agaatcctgg
     3841 cccagcggcc gcaaccatgg tgagcaaggg cgaggaggat aacatggcca tcatcaagga
     3901 gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac ggccacgagt tcgagatcga
     3961 gggcgagggc gagggccgcc cctacgaggg cacccagacc gccaagctga aggtgaccaa
     4021 gggtggcccc ctgcccttcg cctgggacat cctgtcccct cagttcatgt acggctccaa
     4081 ggcctacgtg aagcaccccg ccgacatccc cgactacttg aagctgtcct tccccgaggg
     4141 cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc gtggtgaccg tgacccagga
     4201 ctcctctctc caggacggcg agttcatcta caaggtgaag ctgcgcggca ccaacttccc
     4261 ctccgacggc cccgtaatgc agaagaagac catgggctgg gaggcctcct ccgagcggat
     4321 gtaccccgag gacggcgctc tgaaaggaga gatcaagcag aggctgaagc tgaaggacgg
     4381 cggccactac gacgctgagg tcaagaccac ctacaaggcc aagaagcccg tgcagctgcc
     4441 cggcgcctac aacgtcaaca tcaagttgga catcacctcc cacaacgagg actacaccat
     4501 cgtggaacag tacgaacgcg ccgagggacg gcactccacc ggaggaatgg acgagctgta
     4561 caagtaagcc tgcaggaatt gcctaggaac tttattggac tcgagaagaa ggtatattgc
     4621 tgttgaccca gagcgttggg caggtctgaa gttcattagt gaagccacag atgtaatgaa
     4681 cttcaggacc tgcccactgc cttgggcctc ggacttcaag gggtcagtca cactctttcc
     4741 ctacacgacg ctcttccgat cttgttcttc tatctagagt tgaaccgact agtaagccag
     4801 gtcgagacat gtttaagggt tccggttcca ctaggtacaa ttcgatatca agcttatcga
     4861 taatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     4921 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcta ttgcttcccg
     4981 tatggctttc attttctcct ccttgtataa atcctggttg ctgtctcttt atgaggagtt
     5041 gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg tttgctgacg caacccccac
     5101 tggttggggc attgccacca cctgtcagct cctttccggg actttcgctt tccccctccc
     5161 tattgccacg gcggaactca tcgccgcctg ccttgcccgc tgctggacag gggctcggct
     5221 gttgggcact gacaattccg tggtgttgtc ggggaaatca tcgtcctttc cttggctgct
     5281 cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc tgctacgtcc cttcggccct
     5341 caatccagcg gaccttcctt cccgcggcct gctgccggct ctgcggcctc ttccgcgtct
     5401 tcgccttcgc cctcagacga gtcggatctc cctttgggcc gcctccccgc atcgataccg
     5461 tcgacctcga tcgagaccta gaaaaacatg gagcaatcac aagtagcaat acagcagcta
     5521 ccaatgctga ttgtgcctgg ctagaagcac aagaggagga ggaggtgggt tttccagtca
     5581 cacctcaggt acctttaaga ccaatgactt acaaggcagc tgtagatctt agccactttt
     5641 taaaagaaaa ggggggactg gaagggctaa ttcactccca acgaagacaa gatatccttg
     5701 atctgtggat ctaccacaca caaggctact tccctgattg gcagaactac acaccagggc
     5761 cagggatcag atatccactg acctttggat ggtgctacaa gctagtacca gttgagcaag
     5821 agaaggtaga agaagccaat gaaggagaga acacccgctt gttacaccct gtgagcctgc
     5881 atgggatgga tgacccggag agagaagtat tagagtggag gtttgacagc cgcctagcat
     5941 ttcatcacat ggcccgagag ctgcatccgg actgtactgg gtctctctgg ttagaccaga
     6001 tctgagcctg ggagctctct ggctaactag ggaacccact gcttaagcct caataaagct
     6061 tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg tgactctggt aactagagat
     6121 ccctcagacc cttttagtca gtgtggaaaa tctctagcag catgtgagca aaaggccagc
     6181 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     6241 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     6301 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     6361 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     6421 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     6481 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     6541 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     6601 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6661 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6721 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6781 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6841 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6901 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6961 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     7021 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     7081 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     7141 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     7201 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     7261 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     7321 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     7381 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     7441 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     7501 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     7561 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     7621 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7681 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7741 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7801 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7861 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7921 aaaataaaca aataggggtt ccgcgcacat ttc
//
