LOCUS       sequence-374769-        9574 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral sgRNA vector for Perturb-seq with mU6 sgRNA promoter, CR1
            constant region with CS1 capture sequence in stem loop, and UCOE 
            EF1alpha driving PURO-GFP marker expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9574)
  AUTHORS   Replogle JM, Bonnar JL, Pogson AN, Liem CR, Maier NK, Ding Y, 
            Russell BJ, Wang X, Leng K, Guna A, Norman TM, Pak RA, Ramos DM, 
            Ward ME, Gilbert LA, Kampmann M, Weissman JS, Jost M
  TITLE     Maximizing CRISPRi efficacy and accessibility with dual-sgRNA 
            libraries and optimal effectors.
  JOURNAL   eLife. 2022 Dec 28;11:e81856. doi: 10.7554/eLife.81856.
  PUBMED    36576240
REFERENCE   2  (bases 1 to 9574)
  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"; journalName: "eLife."; 
            date: "2022-12-28"; volume: "11:e81856. doi"; pages: " 
            10.7554/eLife.81856"
COMMENT     Sequence Label: pJR101
FEATURES             Location/Qualifiers
     source          1..9574
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(86..105)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        277..656
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        658..856
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     811..831
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             874..1054
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1101..1226
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1723..1956
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2141..2185
                     /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    2483..2600
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2853..2886)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2890..2910
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        3812..4990
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4043..4981
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4938..4958
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     complement(5002..5021)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             5608..5661
                     /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"
     protein_bind    complement(6413..6446)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    6502..7090
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6555..6575)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6973..6984)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7093..7109)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7094..7110)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             7619..7799
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7861..8449)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7941..7960)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8620..9411,9412..9480))
                     /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     9243..9262
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(9481..9574,1..11))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
       61 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
      121 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
      181 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
      241 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
      301 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      361 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      421 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      481 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      541 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      601 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
      661 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
      721 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
      781 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
      841 aggtctatat aagcagcgcg ttttgcctgt actgggtctc tctggttaga ccagatctga
      901 gcctgggagc tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct
      961 tgagtgcttc aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc
     1021 agaccctttt agtcagtgtg gaaaatctct agcagtggcg cccgaacagg gacttgaaag
     1081 cgaaagggaa accagaggag ctctctcgac gcaggactcg gcttgctgaa gcgcgcacgg
     1141 caagaggcga ggggcggcga ctggtgagta cgccaaaaat tttgactagc ggaggctaga
     1201 aggagagaga tgggtgcgag agcgtcagta ttaagcgggg gagaattaga tcgcgatggg
     1261 aaaaaattcg gttaaggcca gggggaaaga aaaaatataa attaaaacat atagtatggg
     1321 caagcaggga gctagaacga ttcgcagtta atcctggcct gttagaaaca tcagaaggct
     1381 gtagacaaat actgggacag ctacaaccat cccttcagac aggatcagaa gaacttagat
     1441 cattatataa tacagtagca accctctatt gtgtgcatca aaggatagag ataaaagaca
     1501 ccaaggaagc tttagacaag atagaggaag agcaaaacaa aagtaagacc accgcacagc
     1561 aagcggccgg ccgctgatct tcagacctgg aggaggagat atgagggaca attggagaag
     1621 tgaattatat aaatataaag tagtaaaaat tgaaccatta ggagtagcac ccaccaaggc
     1681 aaagagaaga gtggtgcaga gagaaaaaag agcagtggga ataggagctt tgttccttgg
     1741 gttcttggga gcagcaggaa gcactatggg cgcagcgtca atgacgctga cggtacaggc
     1801 cagacaatta ttgtctggta tagtgcagca gcagaacaat ttgctgaggg ctattgaggc
     1861 gcaacagcat ctgttgcaac tcacagtctg gggcatcaag cagctccagg caagaatcct
     1921 ggctgtggaa agatacctaa aggatcaaca gctcctgggg atttggggtt gctctggaaa
     1981 actcatttgc accactgctg tgccttggaa tgctagttgg agtaataaat ctctggaaca
     2041 gatttggaat cacacgacct ggatggagtg ggacagagaa attaacaatt acacaagctt
     2101 aatacactcc ttaattgaag aatcgcaaaa ccagcaagaa aagaatgaac aagaattatt
     2161 ggaattagat aaatgggcaa gtttgtggaa ttggtttaac ataacaaatt ggctgtggta
     2221 tataaaatta ttcataatga tagtaggagg cttggtaggt ttaagaatag tttttgctgt
     2281 actttctata gtgaatagag ttaggcaggg atattcacca ttatcgtttc agacccacct
     2341 cccaaccccg aggggacccg acaggcccga aggaatagaa gaagaaggtg gagagagaga
     2401 cagagacaga tccattcgat tagtgaacgg atcggcactg cgtgcgccaa ttctgcagac
     2461 aaatggcagt attcatccac aattttaaaa gaaaaggggg gattgggggg tacagtgcag
     2521 gggaaagaat agtagacata atagcaacag acatacaaac taaagaatta caaaaacaaa
     2581 ttacaaaaat tcaaaatttt cgggtttatt acagggacag cagagatcca gtttggttag
     2641 taccgggccc gctctagaga tccgacgcgc catctctagg cccgcgccgg ccccctcgca
     2701 cggacttgtg ggagaagctc ggctactccc ctgccccggt taatttgcat ataatatttc
     2761 ctagtaacta tagaggctta atgtgcgata aaagacagat aatctgttct ttttaatact
     2821 agctacattt tacatgatag gcttggattt ctataacttc gtatagcata cattatacga
     2881 agttataaac agcacaaaag gaaactcacc ctaactgtaa agtaattgtg tgttttgaga
     2941 ctataagtat cccttggaga accaccttgt tgggcgagca cagctaaggc cagtttaaga
     3001 gctaagctgg aaacagcata gcaagtttaa ataaggctag tccgttatca acttggccgc
     3061 tttaaggccg gtcctagcaa ggccaagtgg caccgagtcg gtgctttttt tctcgagtac
     3121 taggatccat taggcggccg ccgggaggtg gtccctgcag ttacgccaat gataaccccc
     3181 gccagaaaaa tcttagtagc cttccctttt tgttttccgt gccccaactc ggcggattga
     3241 ctcggcccct tccggaaaca cccgaatcaa cttctagtca aattattgtt cacgccgcaa
     3301 tgacccaccc ctggcccgcg tctgtggaac tgacccctgg tgtacaggag agttcgctgc
     3361 tgaaagtggt cccaaagggg tactagtttt taagctccca actccccctc ccccagcgtc
     3421 tggaggattc cacaccctcg caccgcaggg gcgaggaagt gggcggagtc cggttttggc
     3481 gccagccgct gaggctgcca agcagaaaag ccaccgctga ggagactccg gtcactgtcc
     3541 tcgccccgcc tcccccttcc ctccccttgg ggaccaccgg gcgccacgcc gcgaacggta
     3601 agtgccgcgg tcgtcggcgc ctccgccctc cccctagggc cccaattccc agcgggcgcg
     3661 gcgcgcggcc cctccccccg ccgggcgcgc gcccgctgcc ccgcccttcg tggccgcccg
     3721 gcgtgggcgg tgccacccct ccccccggcg gccccgcgcg cagctcccgg ctccctcccc
     3781 cttcggatgt ggcttgagct gtaggcgcgg agatgcatgt gcccgtcagt gggcagagcg
     3841 cacatcgccc acagtccccg agaagttggg gggaggggtc ggcaattgaa ccggtgccta
     3901 gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc gcctttttcc
     3961 cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa
     4021 cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt
     4081 tacgggttat ggcccttgcg tgccttgaat tacttccacc tggctgcagt acgtgattct
     4141 tgatcccgag cttcgggttg gaagtgggtg ggagagttcg aggccttgcg cttaaggagc
     4201 cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat
     4261 ctggtggcac cttcgcgcct gtctcgctgc tttcgataag tctctagcca tttaaaattt
     4321 ttgatgacct gctgcgacgc tttttttctg gcaagatagt cttgtaaatg cgggccaaga
     4381 tctgcacact ggtatttcgg tttttggggc cgcgggcggc gacggggccc gtgcgtccca
     4441 gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca ccgagaatcg gacgggggta
     4501 gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc
     4561 ctgggcggca aggctggccc ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc
     4621 cggccctgct gcagggagct caaaatggag gacgcggcgc tcgggagagc gggcgggtga
     4681 gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc gtcgcttcat gtgactccac
     4741 ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg cgcttttgga gtacgtcgtc
     4801 tttaggttgg ggggaggggt tttatgcgat ggagtttccc cacactgagt gggtggagac
     4861 tgaagttagg ccagcttggc acttgatgta attctccttg gaatttgccc tttttgagtt
     4921 tggatcttgg ttcattctca agcctcagac agtggttcaa agtttttttc ttccatttca
     4981 ggtgtcgtga gctagcccac catgaccgag tacaagccca ccgttcgcct ggccacaaga
     5041 gatgacgtgc ctcgcgccgt gagaactttg gctgcggcgt tcgccgatta tccagcaacc
     5101 aggcacacag tggaccctga tagacacatc gagcgagtga ctgagctcca ggaactgttt
     5161 ctgacacggg ttggtctgga tatcggaaaa gtgtgggtcg ctgacgatgg ggccgccgtg
     5221 gcagtctgga caaccccaga atccgtggag gccggagcag tattcgccga gataggcccc
     5281 cgcatggcgg agctttcggg gtcaaggctg gctgcacagc agcaaatgga gggcctactc
     5341 gctccacacc ggcctaaaga acccgcctgg ttcctagcca ctgttggcgt gtcaccggac
     5401 catcagggaa aagggctggg ctctgctgtg gtgctgcctg gtgtcgaggc tgcggagagg
     5461 gccggagtgc cggcttttct tgaaactagc gcacctagga acttaccttt ctacgagcgt
     5521 ctgggattta ccgtcacagc cgacgtggag gttccagagg ggccccggac ttggtgtatg
     5581 acacggaaac cgggcgcagg ttctggggag ggtagggggt cacttctgac gtgcggggat
     5641 gtggaagaaa accccggccc accagtagca acgatggtca gtaagggcga ggaacttttt
     5701 acaggggtcg ttcccattct ggttgaattg gatggcgatg tgaacggaca taaatttagc
     5761 gtgtccggcg aaggagaagg tgatgctacc tacggaaagc ttacgttgaa gtttatttgc
     5821 acgaccggta agctgcccgt accatggcca acattggtga ccactctgac ctatggagtg
     5881 cagtgtttca gccggtaccc cgaccacatg aaacaacacg acttcttcaa gtctgccatg
     5941 cctgaaggtt atgtccagga acgtaccatc ttttttaaag acgacggaaa ttataagacc
     6001 cgagccgagg taaagtttga gggcgacact ctcgtcaacc gcatagaatt gaaggggatt
     6061 gacttcaaag aagatggcaa cattctaggc cataaattag agtataatta caacagccac
     6121 aacgtttaca tcatggctga caaacagaag aatggcatca aggcaaattt caagataaga
     6181 cacaatatcg aggacgggag tgtccaactg gccgaccatt atcagcagaa tacacctatt
     6241 ggcgacggac ctgtcctcct ccccgacaat cattacctga gtactcaatc cgcactctcc
     6301 aaagatccca acgagaagag agatcacatg gtattactgg agttcgtgac agctgccggg
     6361 atcaccctcg gcatggatga actctacaag tgagaattcg tcgagggacc taataacttc
     6421 gtatagcata cattatacga agttatacat gtttaagggt tccggttcca ctaggtacaa
     6481 ttcgatatca agcttatcga taatcaacct ctggattaca aaatttgtga aagattgact
     6541 ggtattctta actatgttgc tccttttacg ctatgtggat acgctgcttt aatgcctttg
     6601 tatcatgcta ttgcttcccg tatggctttc attttctcct ccttgtataa atcctggttg
     6661 ctgtctcttt atgaggagtt gtggcccgtt gtcaggcaac gtggcgtggt gtgcactgtg
     6721 tttgctgacg caacccccac tggttggggc attgccacca cctgtcagct cctttccggg
     6781 actttcgctt tccccctccc tattgccacg gcggaactca tcgccgcctg ccttgcccgc
     6841 tgctggacag gggctcggct gttgggcact gacaattccg tggtgttgtc ggggaaatca
     6901 tcgtcctttc cttggctgct cgcctgtgtt gccacctgga ttctgcgcgg gacgtccttc
     6961 tgctacgtcc cttcggccct caatccagcg gaccttcctt cccgcggcct gctgccggct
     7021 ctgcggcctc ttccgcgtct tcgccttcgc cctcagacga gtcggatctc cctttgggcc
     7081 gcctccccgc atcgataccg tcgacctcga tcgagaccta gaaaaacatg gagcaatcac
     7141 aagtagcaat acagcagcta ccaatgctga ttgtgcctgg ctagaagcac aagaggagga
     7201 ggaggtgggt tttccagtca cacctcaggt acctttaaga ccaatgactt acaaggcagc
     7261 tgtagatctt agccactttt taaaagaaaa ggggggactg gaagggctaa ttcactccca
     7321 acgaagacaa gatatccttg atctgtggat ctaccacaca caaggctact tccctgattg
     7381 gcagaactac acaccagggc cagggatcag atatccactg acctttggat ggtgctacaa
     7441 gctagtacca gttgagcaag agaaggtaga agaagccaat gaaggagaga acacccgctt
     7501 gttacaccct gtgagcctgc atgggatgga tgacccggag agagaagtat tagagtggag
     7561 gtttgacagc cgcctagcat ttcatcacat ggcccgagag ctgcatccgg actgtactgg
     7621 gtctctctgg ttagaccaga tctgagcctg ggagctctct ggctaactag ggaacccact
     7681 gcttaagcct caataaagct tgccttgagt gcttcaagta gtgtgtgccc gtctgttgtg
     7741 tgactctggt aactagagat ccctcagacc cttttagtca gtgtggaaaa tctctagcag
     7801 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     7861 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     7921 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     7981 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     8041 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     8101 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     8161 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     8221 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     8281 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     8341 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     8401 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     8461 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     8521 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     8581 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     8641 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     8701 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     8761 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     8821 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     8881 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     8941 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     9001 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     9061 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     9121 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     9181 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     9241 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     9301 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     9361 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     9421 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     9481 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     9541 catatttgaa tgtatttaga aaaataaaca aata
//
