LOCUS       sequence-161846-        7955 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral expression of shRNAs, compatible with double-shRNA 
            expression, SFFV promoter, BFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7955)
  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 7955)
  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: pMK1224
FEATURES             Location/Qualifiers
     source          1..7955
                     /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..4549
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="MSELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKV
                     VEGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     TQDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALK
                     LVGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYC
                     DLPSKLGHKLN"
     ncRNA           4641..4655
                     /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    4844..5432
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(4897..4917)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(5315..5326)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(5435..5451)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5436..5452)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             5961..6141
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(6203..6791)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6283..6302)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6962..7753,7754..7822))
                     /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     7585..7604
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7823..7927)
                     /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 cgccaccatg agcgagctga ttaaggagaa catgcacatg aagctgtaca tggagggcac
     3901 cgtggacaac catcacttca agtgcacatc cgagggcgaa ggcaagccct acgagggcac
     3961 ccagaccatg agaatcaagg tggtcgaggg cggccctctc cccttcgctt tcgacatcct
     4021 ggctactagc ttcctctacg gcagcaagac cttcatcaac cacacacagg gcatccccga
     4081 cttcttcaag cagtccttcc ctgagggctt cacatgggag agagtcacca catacgaaga
     4141 cgggggcgtg ctgaccgcta cccaggacac cagcctccag gacggctgcc tcatctacaa
     4201 cgtcaagatc agaggggtga acttcacatc caacggccct gtgatgcaga agaaaacact
     4261 cggctgggag gccttcaccg agacgctgta ccccgctgac ggcggcctgg aaggcagaaa
     4321 cgacatggcc ctgaagctcg tgggcgggag ccatctgatc gcaaacatca agaccacata
     4381 tagatccaag aaacccgcta agaacctcaa gatgcctggc gtctactatg tggactacag
     4441 actggaaaga atcaaggagg ccaacaacga gacctacgtc gagcagcacg aggtggcagt
     4501 ggccagatac tgcgacctcc ctagcaaact ggggcacaaa cttaattaag cctgcaggaa
     4561 ttgcctagga tgagtatctg actcgagaag aaggtatatt gctgttgacc cagagcgttg
     4621 ggcaggtctg aagttcatta gtgaagccac agatgtaatg aacttcagga cctgcccact
     4681 gccttgggcc tcggacttca aggggtcagt cacactcttt ccctacacga cgctcttccg
     4741 atctctacat tcgctctaga gttgaaccga ctagtaagcc aggtcgagac atgtttaagg
     4801 gttccggttc cactaggtac aattcgatat caagcttatc gataatcaac ctctggatta
     4861 caaaatttgt gaaagattga ctggtattct taactatgtt gctcctttta cgctatgtgg
     4921 atacgctgct ttaatgcctt tgtatcatgc tattgcttcc cgtatggctt tcattttctc
     4981 ctccttgtat aaatcctggt tgctgtctct ttatgaggag ttgtggcccg ttgtcaggca
     5041 acgtggcgtg gtgtgcactg tgtttgctga cgcaaccccc actggttggg gcattgccac
     5101 cacctgtcag ctcctttccg ggactttcgc tttccccctc cctattgcca cggcggaact
     5161 catcgccgcc tgccttgccc gctgctggac aggggctcgg ctgttgggca ctgacaattc
     5221 cgtggtgttg tcggggaaat catcgtcctt tccttggctg ctcgcctgtg ttgccacctg
     5281 gattctgcgc gggacgtcct tctgctacgt cccttcggcc ctcaatccag cggaccttcc
     5341 ttcccgcggc ctgctgccgg ctctgcggcc tcttccgcgt cttcgccttc gccctcagac
     5401 gagtcggatc tccctttggg ccgcctcccc gcatcgatac cgtcgacctc gatcgagacc
     5461 tagaaaaaca tggagcaatc acaagtagca atacagcagc taccaatgct gattgtgcct
     5521 ggctagaagc acaagaggag gaggaggtgg gttttccagt cacacctcag gtacctttaa
     5581 gaccaatgac ttacaaggca gctgtagatc ttagccactt tttaaaagaa aaggggggac
     5641 tggaagggct aattcactcc caacgaagac aagatatcct tgatctgtgg atctaccaca
     5701 cacaaggcta cttccctgat tggcagaact acacaccagg gccagggatc agatatccac
     5761 tgacctttgg atggtgctac aagctagtac cagttgagca agagaaggta gaagaagcca
     5821 atgaaggaga gaacacccgc ttgttacacc ctgtgagcct gcatgggatg gatgacccgg
     5881 agagagaagt attagagtgg aggtttgaca gccgcctagc atttcatcac atggcccgag
     5941 agctgcatcc ggactgtact gggtctctct ggttagacca gatctgagcc tgggagctct
     6001 ctggctaact agggaaccca ctgcttaagc ctcaataaag cttgccttga gtgcttcaag
     6061 tagtgtgtgc ccgtctgttg tgtgactctg gtaactagag atccctcaga cccttttagt
     6121 cagtgtggaa aatctctagc agcatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     6181 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     6241 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     6301 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     6361 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     6421 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     6481 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     6541 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     6601 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     6661 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     6721 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     6781 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     6841 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     6901 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     6961 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     7021 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     7081 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     7141 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     7201 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     7261 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     7321 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     7381 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     7441 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     7501 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     7561 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     7621 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     7681 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     7741 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     7801 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     7861 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     7921 ttccgcgcac atttccccga aaagtgccac ctgac
//
