LOCUS       sequence-161844-        7964 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 7964)
  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 7964)
  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..7964
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(56..75)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        247..626
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        628..826
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     781..801
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             844..1024
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1071..1196
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1695..1928
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2113..2157
                     /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    2455..2572
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     promoter        2757..3164
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     CDS             3176..3772
                     /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(3176..3195)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     3672..3692
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             3782..3835
                     /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             3848..4558
                     /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(3993..4011)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4261..4280
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     ncRNA           4650..4664
                     /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    4853..5441
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4906..4926)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5324..5335)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5444..5460)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5445..5461)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5970..6150
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6212..6800)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6292..6311)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6971..7762,7763..7831))
                     /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     7594..7613
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7832..7936)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gcttaagcgg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
       61 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
      121 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
      181 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata
      241 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
      301 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      361 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      421 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      481 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      541 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      601 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      661 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      721 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      781 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagcgcg ttttgcctgt
      841 actgggtctc tctggttaga ccagatctga gcctgggagc tctctggcta actagggaac
      901 ccactgctta agcctcaata aagcttgcct tgagtgcttc aagtagtgtg tgcccgtctg
      961 ttgtgtgact ctggtaacta gagatccctc agaccctttt agtcagtgtg gaaaatctct
     1021 agcagtggcg cccgaacagg gacttgaaag cgaaagggaa accagaggag ctctctcgac
     1081 gcaggactcg gcttgctgaa gcgcgcacgg caagaggcga ggggcggcga ctggtgagta
     1141 cgccaaaaat tttgactagc ggaggctaga aggagagaga tgggtgcgag agcgtcagta
     1201 ttaagcgggg gagaattaga tcgcgatggg aaaaaattcg gttaaggcca gggggaaaga
     1261 aaaaatataa attaaaacat atagtatggg caagcaggga gctagaacga ttcgcagtta
     1321 atcctggcct gttagaaaca tcagaaggct gtagacaaat actgggacag ctacaaccat
     1381 cccttcagac aggatcagaa gaacttagat cattatataa tacagtagca accctctatt
     1441 gtgtgcatca aaggatagag ataaaagaca ccaaggaagc tttagacaag atagaggaag
     1501 agcaaaacaa aagtaagacc accgcacagc aagcggccgg ccgcgctgat cttcagacct
     1561 ggaggaggag atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa
     1621 attgaaccat taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa
     1681 agagcagtgg gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg
     1741 ggcgcagcgt caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag
     1801 cagcagaaca atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc
     1861 tggggcatca agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa
     1921 cagctcctgg ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg
     1981 aatgctagtt ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag
     2041 tgggacagag aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa
     2101 aaccagcaag aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg
     2161 aattggttta acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga
     2221 ggcttggtag gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag
     2281 ggatattcac cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc
     2341 gaaggaatag aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac
     2401 ggatcggcac tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa
     2461 aagaaaaggg gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac
     2521 agacatacaa actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta
     2581 ttacagggac agcagagatc cagtttggtt agtaccgggc ccgctctagg gccgcgtgga
     2641 taaccgtatt accgccatgc attcctgcag ccccgataaa ataaaagatt ttatttagtc
     2701 tccagaaaaa ggggggaatg aaagacccca cctgtaggtt tggcaagcta gctgcagtaa
     2761 cgccattttg caaggcatgg aaaaatacca aaccaagaat agagaagttc agatcaaggg
     2821 cgggtacatg aaaatagcta acgttgggcc aaacaggata tctgcggtga gcagtttcgg
     2881 ccccggcccg gggccaagaa cagatggtca ccgcagtttc ggccccggcc cgaggccaag
     2941 aacagatggt ccccagatat ggcccaaccc tcagcagttt cttaagaccc atcagatgtt
     3001 tccaggctcc cccaaggacc tgaaatgacc ctgcgcctta tttgaattaa ccaatcagcc
     3061 tgcttctcgc ttctgttcgc gcgcttctgc ttcccgagct ctataaaaga gctcacaacc
     3121 cctcactcgg cgcgccagtc ctccgacaga ctgagtcgcc cgggtctagc ccaccatgac
     3181 cgagtacaag cccacggtgc gcctcgccac ccgcgacgac gtcccccggg ccgtacgcac
     3241 cctcgccgcc gcgttcgccg actaccccgc cacgcgccac accgtcgacc cggaccgcca
     3301 catcgagcgg gtcaccgagc tgcaagaact cttcctcacg cgcgtcgggc tcgacatcgg
     3361 caaggtgtgg gtcgcggacg acggcgccgc ggtggcggtc tggaccacgc cggagagcgt
     3421 cgaagcgggg gcggtgttcg ccgagatcgg cccgcgcatg gccgagttga gcggttcccg
     3481 gctggccgcg cagcaacaga tggaaggcct cctggcgccg caccggccca aggagcccgc
     3541 gtggttcctg gccaccgtcg gcgtctcgcc cgaccaccag ggcaagggtc tgggcagcgc
     3601 cgtcgtgctc cccggagtgg aggcggccga gcgcgccggg gtgcccgcct tcctggagac
     3661 ctccgcgccc cgcaacctcc ccttctacga gcggctcggc ttcaccgtca ccgccgacgt
     3721 cgaggtgccc gaaggaccgc gcacctggtg catgacccgc aagcccggtg ccggatcggg
     3781 agagggcaga ggaagtctgc taacatgcgg tgacgtcgag gagaatcctg gcccagcggc
     3841 cgcaaccatg gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg
     3901 cttcaaggtg cacatggagg gctccgtgaa cggccacgag ttcgagatcg agggcgaggg
     3961 cgagggccgc ccctacgagg gcacccagac cgccaagctg aaggtgacca agggtggccc
     4021 cctgcccttc gcctgggaca tcctgtcccc tcagttcatg tacggctcca aggcctacgt
     4081 gaagcacccc gccgacatcc ccgactactt gaagctgtcc ttccccgagg gcttcaagtg
     4141 ggagcgcgtg atgaacttcg aggacggcgg cgtggtgacc gtgacccagg actcctctct
     4201 ccaggacggc gagttcatct acaaggtgaa gctgcgcggc accaacttcc cctccgacgg
     4261 ccccgtaatg cagaagaaga ccatgggctg ggaggcctcc tccgagcgga tgtaccccga
     4321 ggacggcgct ctgaaaggag agatcaagca gaggctgaag ctgaaggacg gcggccacta
     4381 cgacgctgag gtcaagacca cctacaaggc caagaagccc gtgcagctgc ccggcgccta
     4441 caacgtcaac atcaagttgg acatcacctc ccacaacgag gactacacca tcgtggaaca
     4501 gtacgaacgc gccgagggac ggcactccac cggaggaatg gacgagctgt acaagtaagc
     4561 ctgcaggaat tgcctaggaa ctttattgga ctcgagaaga aggtatattg ctgttgaccc
     4621 agagcgttgg gcaggtctga agttcattag tgaagccaca gatgtaatga acttcaggac
     4681 ctgcccactg ccttgggcct cggacttcaa ggggtcagtc acactctttc cctacacgac
     4741 gctcttccga tcttgttctt ctatctagag ttgaaccgac tagtaagcca ggtcgagaca
     4801 tgtttaaggg ttccggttcc actaggtaca attcgatatc aagcttatcg ataatcaacc
     4861 tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg ctccttttac
     4921 gctatgtgga tacgctgctt taatgccttt gtatcatgct attgcttccc gtatggcttt
     4981 cattttctcc tccttgtata aatcctggtt gctgtctctt tatgaggagt tgtggcccgt
     5041 tgtcaggcaa cgtggcgtgg tgtgcactgt gtttgctgac gcaaccccca ctggttgggg
     5101 cattgccacc acctgtcagc tcctttccgg gactttcgct ttccccctcc ctattgccac
     5161 ggcggaactc atcgccgcct gccttgcccg ctgctggaca ggggctcggc tgttgggcac
     5221 tgacaattcc gtggtgttgt cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt
     5281 tgccacctgg attctgcgcg ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc
     5341 ggaccttcct tcccgcggcc tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg
     5401 ccctcagacg agtcggatct ccctttgggc cgcctccccg catcgatacc gtcgacctcg
     5461 atcgagacct agaaaaacat ggagcaatca caagtagcaa tacagcagct accaatgctg
     5521 attgtgcctg gctagaagca caagaggagg aggaggtggg ttttccagtc acacctcagg
     5581 tacctttaag accaatgact tacaaggcag ctgtagatct tagccacttt ttaaaagaaa
     5641 aggggggact ggaagggcta attcactccc aacgaagaca agatatcctt gatctgtgga
     5701 tctaccacac acaaggctac ttccctgatt ggcagaacta cacaccaggg ccagggatca
     5761 gatatccact gacctttgga tggtgctaca agctagtacc agttgagcaa gagaaggtag
     5821 aagaagccaa tgaaggagag aacacccgct tgttacaccc tgtgagcctg catgggatgg
     5881 atgacccgga gagagaagta ttagagtgga ggtttgacag ccgcctagca tttcatcaca
     5941 tggcccgaga gctgcatccg gactgtactg ggtctctctg gttagaccag atctgagcct
     6001 gggagctctc tggctaacta gggaacccac tgcttaagcc tcaataaagc ttgccttgag
     6061 tgcttcaagt agtgtgtgcc cgtctgttgt gtgactctgg taactagaga tccctcagac
     6121 ccttttagtc agtgtggaaa atctctagca gcatgtgagc aaaaggccag caaaaggcca
     6181 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     6241 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     6301 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     6361 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6421 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6481 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6541 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6601 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     6661 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6721 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6781 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6841 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6901 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6961 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     7021 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     7081 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     7141 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     7201 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     7261 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     7321 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     7381 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7441 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7501 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     7561 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     7621 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7681 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7741 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7801 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7861 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7921 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgac
//
