LOCUS       sequence-387135-        8828 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Cassette used in dual sgRNA library generation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8828)
  AUTHORS   Guna A, Page KR, Replogle JM, Esantsi TK, Wang ML, Weissman JS, 
            Voorhees RM
  TITLE     A dual sgRNA library design to probe genetic modifiers using 
            genome-wide CRISPRi screens.
  JOURNAL   BMC Genomics. 2023 Oct 30;24(1):651. doi: 
            10.1186/s12864-023-09754-y.
  PUBMED    37904134
REFERENCE   2  (bases 1 to 8828)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "BMC 
            Genomics."; date: "2023-10-30"; volume: "24(1):651. doi"; pages: " 
            10.1186/s12864-023-09754-y"
COMMENT     Sequence Label: pJR152
FEATURES             Location/Qualifiers
     source          1..8828
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1211..1230)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1402..1781
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1783..1981
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1936..1956
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             1999..2179
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    2226..2351
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    2848..3081
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             3266..3310
                     /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    3608..3725
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     3813..3833
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3984..4003
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        4231..4442
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          4443..5381
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     5338..5358
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             5402..5998
                     /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(5402..5421)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     5898..5918
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             6008..6061
                     /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             6074..6772
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(6792..6825)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    6881..7469
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(6934..6954)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(7352..7363)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(7472..7488)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7473..7489)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             7998..8178
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(8240..8828)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(8320..8339)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     1201 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     1261 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     1321 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgcgatg
     1381 tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag taatcaatta
     1441 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
     1501 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
     1561 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
     1621 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     1681 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     1741 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     1801 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1861 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     1921 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     1981 gcgcgttttg cctgtactgg gtctctctgg ttagaccaga tctgagcctg ggagctctct
     2041 ggctaactag ggaacccact gcttaagcct caataaagct tgccttgagt gcttcaagta
     2101 gtgtgtgccc gtctgttgtg tgactctggt aactagagat ccctcagacc cttttagtca
     2161 gtgtggaaaa tctctagcag tggcgcccga acagggactt gaaagcgaaa gggaaaccag
     2221 aggagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc
     2281 ggcgactggt gagtacgcca aaaattttga ctagcggagg ctagaaggag agagatgggt
     2341 gcgagagcgt cagtattaag cgggggagaa ttagatcgcg atgggaaaaa attcggttaa
     2401 ggccaggggg aaagaaaaaa tataaattaa aacatatagt atgggcaagc agggagctag
     2461 aacgattcgc agttaatcct ggcctgttag aaacatcaga aggctgtaga caaatactgg
     2521 gacagctaca accatccctt cagacaggat cagaagaact tagatcatta tataatacag
     2581 tagcaaccct ctattgtgtg catcaaagga tagagataaa agacaccaag gaagctttag
     2641 acaagataga ggaagagcaa aacaaaagta agaccaccgc acagcaagcg gccggccgct
     2701 gatcttcaga cctggaggag gagatatgag ggacaattgg agaagtgaat tatataaata
     2761 taaagtagta aaaattgaac cattaggagt agcacccacc aaggcaaaga gaagagtggt
     2821 gcagagagaa aaaagagcag tgggaatagg agctttgttc cttgggttct tgggagcagc
     2881 aggaagcact atgggcgcag cgtcaatgac gctgacggta caggccagac aattattgtc
     2941 tggtatagtg cagcagcaga acaatttgct gagggctatt gaggcgcaac agcatctgtt
     3001 gcaactcaca gtctggggca tcaagcagct ccaggcaaga atcctggctg tggaaagata
     3061 cctaaaggat caacagctcc tggggatttg gggttgctct ggaaaactca tttgcaccac
     3121 tgctgtgcct tggaatgcta gttggagtaa taaatctctg gaacagattt ggaatcacac
     3181 gacctggatg gagtgggaca gagaaattaa caattacaca agcttaatac actccttaat
     3241 tgaagaatcg caaaaccagc aagaaaagaa tgaacaagaa ttattggaat tagataaatg
     3301 ggcaagtttg tggaattggt ttaacataac aaattggctg tggtatataa aattattcat
     3361 aatgatagta ggaggcttgg taggtttaag aatagttttt gctgtacttt ctatagtgaa
     3421 tagagttagg cagggatatt caccattatc gtttcagacc cacctcccaa ccccgagggg
     3481 acccgacagg cccgaaggaa tagaagaaga aggtggagag agagacagag acagatccat
     3541 tcgattagtg aacggatcgg cactgcgtgc gccaattctg cagacaaatg gcagtattca
     3601 tccacaattt taaaagaaaa ggggggattg gggggtacag tgcaggggaa agaatagtag
     3661 acataatagc aacagacata caaactaaag aattacaaaa acaaattaca aaaattcaaa
     3721 attttcgggt ttattacagg gacagcagag atccagtttg gttagtaccg ggcccgctct
     3781 agaggatccc gcgccccaag gtcgggcagg aagagggcct atttcccatg attccttcat
     3841 atttgcatat acgatacaag gctgttagag agataattgg aattaatttg actgtaaaca
     3901 caaagatatt agtacaaaat acgtgacgta gaaagtaata atttcttggg tagtttgcag
     3961 ttttaaaatt atgttttaaa atggactatc atatgcttac cgtaacttga aagtatttcg
     4021 atttcttggc tttatatatc ttgtggaaag ccagaaacat ggaccaggat gggcaccacc
     4081 cgtttcagag ctaagcacaa gagtgcatag caagttgaaa taaggctagt ccgtttacaa
     4141 cttgaaaaag tggcacccga gtcgggtgct ttttttctcg agtgcggccg cgtggataac
     4201 cgtattaccg ccatgcatgt gcccgtcagt gggcagagcg cacatcgccc acagtccccg
     4261 agaagttggg gggaggggtc ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa
     4321 actgggaaag tgatgtcgtg tactggctcc gcctttttcc cgagggtggg ggagaaccgt
     4381 atataagtgc agtagtcgcc gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac
     4441 aggtaagtgc cgtgtgtggt tcccgcgggc ctggcctctt tacgggttat ggcccttgcg
     4501 tgccttgaat tacttccacc tggctgcagt acgtgattct tgatcccgag cttcgggttg
     4561 gaagtgggtg ggagagttcg aggccttgcg cttaaggagc cccttcgcct cgtgcttgag
     4621 ttgaggcctg gcctgggcgc tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct
     4681 gtctcgctgc tttcgataag tctctagcca tttaaaattt ttgatgacct gctgcgacgc
     4741 tttttttctg gcaagatagt cttgtaaatg cgggccaaga tctgcacact ggtatttcgg
     4801 tttttggggc cgcgggcggc gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc
     4861 ggggcctgcg agcgcggcca ccgagaatcg gacgggggta gtctcaagct ggccggcctg
     4921 ctctggtgcc tggcctcgcg ccgccgtgta tcgccccgcc ctgggcggca aggctggccc
     4981 ggtcggcacc agttgcgtga gcggaaagat ggccgcttcc cggccctgct gcagggagct
     5041 caaaatggag gacgcggcgc tcgggagagc gggcgggtga gtcacccaca caaaggaaaa
     5101 gggcctttcc gtcctcagcc gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca
     5161 ggcacctcga ttagttctcg cgcttttgga gtacgtcgtc tttaggttgg ggggaggggt
     5221 tttatgcgat ggagtttccc cacactgagt gggtggagac tgaagttagg ccagcttggc
     5281 acttgatgta attctccttg gaatttgccc tttttgagtt tggatcttgg ttcattctca
     5341 agcctcagac agtggttcaa agtttttttc ttccatttca ggtgtcgtga gctagcccac
     5401 catgaccgag tacaagccca cggtgcgcct cgccacccgc gacgacgtcc cccgggccgt
     5461 acgcaccctc gccgccgcgt tcgccgacta ccccgccacg cgccacaccg tcgacccgga
     5521 ccgccacatc gagcgggtca ccgagctgca agaactcttc ctcacgcgcg tcgggctcga
     5581 catcggcaag gtgtgggtcg cggacgacgg cgccgcggtg gcggtctgga ccacgccgga
     5641 gagcgtcgaa gcgggggcgg tgttcgccga gatcggcccg cgcatggccg agttgagcgg
     5701 ttcccggctg gccgcgcagc aacagatgga aggcctcctg gcgccgcacc ggcccaagga
     5761 gcccgcgtgg ttcctggcca ccgtcggcgt ctcgcccgac caccagggca agggtctggg
     5821 cagcgccgtc gtgctccccg gagtggaggc ggccgagcgc gccggggtgc ccgccttcct
     5881 ggagacctcc gcgccccgca acctcccctt ctacgagcgg ctcggcttca ccgtcaccgc
     5941 cgacgtcgag gtgcccgaag gaccgcgcac ctggtgcatg acccgcaagc ccggtgccgg
     6001 atcgggagag ggcagaggaa gtctgctaac atgcggtgac gtcgaggaga atcctggccc
     6061 accggtcgcc accagcgagc tgattaagga gaacatgcac atgaagctgt acatggaggg
     6121 caccgtggac aaccatcact tcaagtgcac atccgagggc gaaggcaagc cctacgaggg
     6181 cacccagacc atgagaatca aggtggtcga gggcggccct ctccccttcg ccttcgacat
     6241 cctggctact agcttcctct acggcagcaa gaccttcatc aaccacaccc agggcatccc
     6301 cgacttcttc aagcagtcct tccctgaggg cttcacatgg gagagagtca ccacatacga
     6361 agacgggggc gtgctgaccg ctacccagga caccagcctc caggacggct gcctcatcta
     6421 caacgtcaag atcagagggg tgaacttcac atccaacggc cctgtgatgc agaagaaaac
     6481 actcggctgg gaggccttca ccgagacgct gtaccccgct gacggcggcc tggaaggcag
     6541 aaacgacatg gccctgaagc tcgtgggcgg gagccatctg atcgcaaaca tcaagaccac
     6601 atatagatcc aagaaacccg ctaagaacct caagatgcct ggcgtctact atgtggacta
     6661 cagactggaa agaatcaagg aggccaacaa cgagacctac gtcgagcagc acgaggtggc
     6721 agtggccaga tactgcgacc tccctagcaa actggggcac aagcttaatt aagaattcgt
     6781 cgagggacct aataacttcg tatagcatac attatacgaa gttatacatg tttaagggtt
     6841 ccggttccac taggtacaat tcgatatcaa gcttatcgat aatcaacctc tggattacaa
     6901 aatttgtgaa agattgactg gtattcttaa ctatgttgct ccttttacgc tatgtggata
     6961 cgctgcttta atgcctttgt atcatgctat tgcttcccgt atggctttca ttttctcctc
     7021 cttgtataaa tcctggttgc tgtctcttta tgaggagttg tggcccgttg tcaggcaacg
     7081 tggcgtggtg tgcactgtgt ttgctgacgc aacccccact ggttggggca ttgccaccac
     7141 ctgtcagctc ctttccggga ctttcgcttt ccccctccct attgccacgg cggaactcat
     7201 cgccgcctgc cttgcccgct gctggacagg ggctcggctg ttgggcactg acaattccgt
     7261 ggtgttgtcg gggaaatcat cgtcctttcc ttggctgctc gcctgtgttg ccacctggat
     7321 tctgcgcggg acgtccttct gctacgtccc ttcggccctc aatccagcgg accttccttc
     7381 ccgcggcctg ctgccggctc tgcggcctct tccgcgtctt cgccttcgcc ctcagacgag
     7441 tcggatctcc ctttgggccg cctccccgca tcgataccgt cgacctcgat cgagacctag
     7501 aaaaacatgg agcaatcaca agtagcaata cagcagctac caatgctgat tgtgcctggc
     7561 tagaagcaca agaggaggag gaggtgggtt ttccagtcac acctcaggta cctttaagac
     7621 caatgactta caaggcagct gtagatctta gccacttttt aaaagaaaag gggggactgg
     7681 aagggctaat tcactcccaa cgaagacaag atatccttga tctgtggatc taccacacac
     7741 aaggctactt ccctgattgg cagaactaca caccagggcc agggatcaga tatccactga
     7801 cctttggatg gtgctacaag ctagtaccag ttgagcaaga gaaggtagaa gaagccaatg
     7861 aaggagagaa cacccgcttg ttacaccctg tgagcctgca tgggatggat gacccggaga
     7921 gagaagtatt agagtggagg tttgacagcc gcctagcatt tcatcacatg gcccgagagc
     7981 tgcatccgga ctgtactggg tctctctggt tagaccagat ctgagcctgg gagctctctg
     8041 gctaactagg gaacccactg cttaagcctc aataaagctt gccttgagtg cttcaagtag
     8101 tgtgtgcccg tctgttgtgt gactctggta actagagatc cctcagaccc ttttagtcag
     8161 tgtggaaaat ctctagcagc atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     8221 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     8281 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     8341 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     8401 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     8461 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     8521 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     8581 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     8641 agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
     8701 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     8761 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     8821 gatctcaa
//
