LOCUS       sequence-374823-        9548 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral sgRNA vector with mU6 sgRNA promoter, CR1 constant 
            region, and UCOE EF1alpha driving PURO-GFP marker expression.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 9548)
  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 9548)
  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: pJR104
FEATURES             Location/Qualifiers
     source          1..9548
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(77..96)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        268..647
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        649..847
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     802..822
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             865..1045
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1092..1217
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1714..1947
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2132..2176
                     /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    2474..2591
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2844..2877)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2881..2901
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        3777..4955
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          4008..4946
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4903..4923
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     complement(4967..4986)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     CDS             5573..5626
                     /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(6378..6411)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    6467..7055
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6520..6540)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6938..6949)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7058..7074)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7059..7075)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             7584..7764
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7826..8414)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7906..7925)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(8585..9376,9377..9445))
                     /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     9208..9227
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(9446..9548,1..2))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
       61 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
      121 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
      181 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
      241 gcgatgtacg ggccagatat acgcgttgac attgattatt gactagttat taatagtaat
      301 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      361 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      421 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      481 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      541 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
      601 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
      661 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
      721 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
      781 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
      841 taagcagcgc gttttgcctg tactgggtct ctctggttag accagatctg agcctgggag
      901 ctctctggct aactagggaa cccactgctt aagcctcaat aaagcttgcc ttgagtgctt
      961 caagtagtgt gtgcccgtct gttgtgtgac tctggtaact agagatccct cagacccttt
     1021 tagtcagtgt ggaaaatctc tagcagtggc gcccgaacag ggacttgaaa gcgaaaggga
     1081 aaccagagga gctctctcga cgcaggactc ggcttgctga agcgcgcacg gcaagaggcg
     1141 aggggcggcg actggtgagt acgccaaaaa ttttgactag cggaggctag aaggagagag
     1201 atgggtgcga gagcgtcagt attaagcggg ggagaattag atcgcgatgg gaaaaaattc
     1261 ggttaaggcc agggggaaag aaaaaatata aattaaaaca tatagtatgg gcaagcaggg
     1321 agctagaacg attcgcagtt aatcctggcc tgttagaaac atcagaaggc tgtagacaaa
     1381 tactgggaca gctacaacca tcccttcaga caggatcaga agaacttaga tcattatata
     1441 atacagtagc aaccctctat tgtgtgcatc aaaggataga gataaaagac accaaggaag
     1501 ctttagacaa gatagaggaa gagcaaaaca aaagtaagac caccgcacag caagcggccg
     1561 gccgctgatc ttcagacctg gaggaggaga tatgagggac aattggagaa gtgaattata
     1621 taaatataaa gtagtaaaaa ttgaaccatt aggagtagca cccaccaagg caaagagaag
     1681 agtggtgcag agagaaaaaa gagcagtggg aataggagct ttgttccttg ggttcttggg
     1741 agcagcagga agcactatgg gcgcagcgtc aatgacgctg acggtacagg ccagacaatt
     1801 attgtctggt atagtgcagc agcagaacaa tttgctgagg gctattgagg cgcaacagca
     1861 tctgttgcaa ctcacagtct ggggcatcaa gcagctccag gcaagaatcc tggctgtgga
     1921 aagataccta aaggatcaac agctcctggg gatttggggt tgctctggaa aactcatttg
     1981 caccactgct gtgccttgga atgctagttg gagtaataaa tctctggaac agatttggaa
     2041 tcacacgacc tggatggagt gggacagaga aattaacaat tacacaagct taatacactc
     2101 cttaattgaa gaatcgcaaa accagcaaga aaagaatgaa caagaattat tggaattaga
     2161 taaatgggca agtttgtgga attggtttaa cataacaaat tggctgtggt atataaaatt
     2221 attcataatg atagtaggag gcttggtagg tttaagaata gtttttgctg tactttctat
     2281 agtgaataga gttaggcagg gatattcacc attatcgttt cagacccacc tcccaacccc
     2341 gaggggaccc gacaggcccg aaggaataga agaagaaggt ggagagagag acagagacag
     2401 atccattcga ttagtgaacg gatcggcact gcgtgcgcca attctgcaga caaatggcag
     2461 tattcatcca caattttaaa agaaaagggg ggattggggg gtacagtgca ggggaaagaa
     2521 tagtagacat aatagcaaca gacatacaaa ctaaagaatt acaaaaacaa attacaaaaa
     2581 ttcaaaattt tcgggtttat tacagggaca gcagagatcc agtttggtta gtaccgggcc
     2641 cgctctagag atccgacgcg ccatctctag gcccgcgccg gccccctcgc acggacttgt
     2701 gggagaagct cggctactcc cctgccccgg ttaatttgca tataatattt cctagtaact
     2761 atagaggctt aatgtgcgat aaaagacaga taatctgttc tttttaatac tagctacatt
     2821 ttacatgata ggcttggatt tctataactt cgtatagcat acattatacg aagttataaa
     2881 cagcacaaaa ggaaactcac cctaactgta aagtaattgt gtgttttgag actataagta
     2941 tcccttggag aaccaccttg ttggaccagg atgggcacca cccgtttaag agctaagctg
     3001 gaaacagcat agcaagttta aataaggcta gtccgttatc aacttgaaaa agtggcaccg
     3061 agtcggtgct ttttttctcg agtactagga tccattaggc ggccgccggg aggtggtccc
     3121 tgcagttacg ccaatgataa cccccgccag aaaaatctta gtagccttcc ctttttgttt
     3181 tccgtgcccc aactcggcgg attgactcgg ccccttccgg aaacacccga atcaacttct
     3241 agtcaaatta ttgttcacgc cgcaatgacc cacccctggc ccgcgtctgt ggaactgacc
     3301 cctggtgtac aggagagttc gctgctgaaa gtggtcccaa aggggtacta gtttttaagc
     3361 tcccaactcc ccctccccca gcgtctggag gattccacac cctcgcaccg caggggcgag
     3421 gaagtgggcg gagtccggtt ttggcgccag ccgctgaggc tgccaagcag aaaagccacc
     3481 gctgaggaga ctccggtcac tgtcctcgcc ccgcctcccc cttccctccc cttggggacc
     3541 accgggcgcc acgccgcgaa cggtaagtgc cgcggtcgtc ggcgcctccg ccctccccct
     3601 agggccccaa ttcccagcgg gcgcggcgcg cggcccctcc ccccgccggg cgcgcgcccg
     3661 ctgccccgcc cttcgtggcc gcccggcgtg ggcggtgcca cccctccccc cggcggcccc
     3721 gcgcgcagct cccggctccc tcccccttcg gatgtggctt gagctgtagg cgcggagatg
     3781 catgtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag ttggggggag
     3841 gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg gaaagtgatg
     3901 tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata agtgcagtag
     3961 tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta agtgccgtgt
     4021 gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct tgaattactt
     4081 ccacctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga
     4141 gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg
     4201 ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg
     4261 ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag
     4321 atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg
     4381 gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc
     4441 ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc
     4501 tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg
     4561 cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc
     4621 ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct
     4681 cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt
     4741 tctcgcgctt ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt
     4801 ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct
     4861 ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg
     4921 ttcaaagttt ttttcttcca tttcaggtgt cgtgagctag cccaccatga ccgagtacaa
     4981 gcccaccgtt cgcctggcca caagagatga cgtgcctcgc gccgtgagaa ctttggctgc
     5041 ggcgttcgcc gattatccag caaccaggca cacagtggac cctgatagac acatcgagcg
     5101 agtgactgag ctccaggaac tgtttctgac acgggttggt ctggatatcg gaaaagtgtg
     5161 ggtcgctgac gatggggccg ccgtggcagt ctggacaacc ccagaatccg tggaggccgg
     5221 agcagtattc gccgagatag gcccccgcat ggcggagctt tcggggtcaa ggctggctgc
     5281 acagcagcaa atggagggcc tactcgctcc acaccggcct aaagaacccg cctggttcct
     5341 agccactgtt ggcgtgtcac cggaccatca gggaaaaggg ctgggctctg ctgtggtgct
     5401 gcctggtgtc gaggctgcgg agagggccgg agtgccggct tttcttgaaa ctagcgcacc
     5461 taggaactta cctttctacg agcgtctggg atttaccgtc acagccgacg tggaggttcc
     5521 agaggggccc cggacttggt gtatgacacg gaaaccgggc gcaggttctg gggagggtag
     5581 ggggtcactt ctgacgtgcg gggatgtgga agaaaacccc ggcccaccag tagcaacgat
     5641 ggtcagtaag ggcgaggaac tttttacagg ggtcgttccc attctggttg aattggatgg
     5701 cgatgtgaac ggacataaat ttagcgtgtc cggcgaagga gaaggtgatg ctacctacgg
     5761 aaagcttacg ttgaagttta tttgcacgac cggtaagctg cccgtaccat ggccaacatt
     5821 ggtgaccact ctgacctatg gagtgcagtg tttcagccgg taccccgacc acatgaaaca
     5881 acacgacttc ttcaagtctg ccatgcctga aggttatgtc caggaacgta ccatcttttt
     5941 taaagacgac ggaaattata agacccgagc cgaggtaaag tttgagggcg acactctcgt
     6001 caaccgcata gaattgaagg ggattgactt caaagaagat ggcaacattc taggccataa
     6061 attagagtat aattacaaca gccacaacgt ttacatcatg gctgacaaac agaagaatgg
     6121 catcaaggca aatttcaaga taagacacaa tatcgaggac gggagtgtcc aactggccga
     6181 ccattatcag cagaatacac ctattggcga cggacctgtc ctcctccccg acaatcatta
     6241 cctgagtact caatccgcac tctccaaaga tcccaacgag aagagagatc acatggtatt
     6301 actggagttc gtgacagctg ccgggatcac cctcggcatg gatgaactct acaagtgaga
     6361 attcgtcgag ggacctaata acttcgtata gcatacatta tacgaagtta tacatgttta
     6421 agggttccgg ttccactagg tacaattcga tatcaagctt atcgataatc aacctctgga
     6481 ttacaaaatt tgtgaaagat tgactggtat tcttaactat gttgctcctt ttacgctatg
     6541 tggatacgct gctttaatgc ctttgtatca tgctattgct tcccgtatgg ctttcatttt
     6601 ctcctccttg tataaatcct ggttgctgtc tctttatgag gagttgtggc ccgttgtcag
     6661 gcaacgtggc gtggtgtgca ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc
     6721 caccacctgt cagctccttt ccgggacttt cgctttcccc ctccctattg ccacggcgga
     6781 actcatcgcc gcctgccttg cccgctgctg gacaggggct cggctgttgg gcactgacaa
     6841 ttccgtggtg ttgtcgggga aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac
     6901 ctggattctg cgcgggacgt ccttctgcta cgtcccttcg gccctcaatc cagcggacct
     6961 tccttcccgc ggcctgctgc cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca
     7021 gacgagtcgg atctcccttt gggccgcctc cccgcatcga taccgtcgac ctcgatcgag
     7081 acctagaaaa acatggagca atcacaagta gcaatacagc agctaccaat gctgattgtg
     7141 cctggctaga agcacaagag gaggaggagg tgggttttcc agtcacacct caggtacctt
     7201 taagaccaat gacttacaag gcagctgtag atcttagcca ctttttaaaa gaaaaggggg
     7261 gactggaagg gctaattcac tcccaacgaa gacaagatat ccttgatctg tggatctacc
     7321 acacacaagg ctacttccct gattggcaga actacacacc agggccaggg atcagatatc
     7381 cactgacctt tggatggtgc tacaagctag taccagttga gcaagagaag gtagaagaag
     7441 ccaatgaagg agagaacacc cgcttgttac accctgtgag cctgcatggg atggatgacc
     7501 cggagagaga agtattagag tggaggtttg acagccgcct agcatttcat cacatggccc
     7561 gagagctgca tccggactgt actgggtctc tctggttaga ccagatctga gcctgggagc
     7621 tctctggcta actagggaac ccactgctta agcctcaata aagcttgcct tgagtgcttc
     7681 aagtagtgtg tgcccgtctg ttgtgtgact ctggtaacta gagatccctc agaccctttt
     7741 agtcagtgtg gaaaatctct agcagcatgt gagcaaaagg ccagcaaaag gccaggaacc
     7801 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     7861 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     7921 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     7981 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     8041 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     8101 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     8161 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     8221 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     8281 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     8341 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     8401 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     8461 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     8521 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     8581 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     8641 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     8701 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa
     8761 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     8821 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     8881 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     8941 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     9001 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     9061 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     9121 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     9181 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     9241 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     9301 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     9361 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     9421 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     9481 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     9541 gggttccg
//
