LOCUS       sequence-497473-        8903 bp    DNA     circular SYN 15-JAN-2026
DEFINITION  Parental vector for building the dual-sgRNA CRISPRi library. 
            modified hU6-sgRNA library vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8903)
  AUTHORS   Simpson D, Ling J, Jing Y, Adamson B
  TITLE     Mapping the Genetic Interaction Network of PARP inhibitor Response.
  JOURNAL   bioRxiv [Preprint]. 2023 Aug 20:2023.08.19.553986. doi: 
            10.1101/2023.08.19.553986.
  PUBMED    37645833
REFERENCE   2  (bases 1 to 8903)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 15, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2023 Aug 20:2023.08.19.553986. doi: 
            10.1101/2023.08.19.553986."
COMMENT     Sequence Label: pJL052
FEATURES             Location/Qualifiers
     source          1..8903
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(48..67)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        239..618
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        620..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     773..793
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             836..1016
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1063..1188
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1685..1918
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2103..2147
                     /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    2445..2562
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     primer_bind     2702..2722
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2873..2892
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     promoter        3143..3354
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          3355..4293
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4250..4270
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4314..4910
                     /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(4314..4333)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4810..4830
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4920..4973
                     /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             4986..5684
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(5704..5737)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5793..6381
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5846..5866)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6264..6275)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6384..6400)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6385..6401)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6910..7090
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7152..7740)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7232..7251)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7911..8702,8703..8771))
                     /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     8534..8553
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8772..8876)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac aatctgctct
       61 gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt cgctgagtag
      121 tgcgcgagca aaatttaagc tacaacaagg caaggcttga ccgacaattg catgaagaat
      181 ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac gggccagata tacgcgttga
      241 cattgattat tgactagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca
      301 tatatggagt tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac
      361 gacccccgcc cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact
      421 ttccattgac gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa
      481 gtgtatcata tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg
      541 cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta
      601 gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg
      661 tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg
      721 caccaaaatc aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg
      781 ggcggtaggc gtgtacggtg ggaggtctat ataagcagcg cgttttgcct gtactgggtc
      841 tctctggtta gaccagatct gagcctggga gctctctggc taactaggga acccactgct
      901 taagcctcaa taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtga
      961 ctctggtaac tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagtgg
     1021 cgcccgaaca gggacttgaa agcgaaaggg aaaccagagg agctctctcg acgcaggact
     1081 cggcttgctg aagcgcgcac ggcaagaggc gaggggcggc gactggtgag tacgccaaaa
     1141 attttgacta gcggaggcta gaaggagaga gatgggtgcg agagcgtcag tattaagcgg
     1201 gggagaatta gatcgcgatg ggaaaaaatt cggttaaggc cagggggaaa gaaaaaatat
     1261 aaattaaaac atatagtatg ggcaagcagg gagctagaac gattcgcagt taatcctggc
     1321 ctgttagaaa catcagaagg ctgtagacaa atactgggac agctacaacc atcccttcag
     1381 acaggatcag aagaacttag atcattatat aatacagtag caaccctcta ttgtgtgcat
     1441 caaaggatag agataaaaga caccaaggaa gctttagaca agatagagga agagcaaaac
     1501 aaaagtaaga ccaccgcaca gcaagcggcc ggccgctgat cttcagacct ggaggaggag
     1561 atatgaggga caattggaga agtgaattat ataaatataa agtagtaaaa attgaaccat
     1621 taggagtagc acccaccaag gcaaagagaa gagtggtgca gagagaaaaa agagcagtgg
     1681 gaataggagc tttgttcctt gggttcttgg gagcagcagg aagcactatg ggcgcagcgt
     1741 caatgacgct gacggtacag gccagacaat tattgtctgg tatagtgcag cagcagaaca
     1801 atttgctgag ggctattgag gcgcaacagc atctgttgca actcacagtc tggggcatca
     1861 agcagctcca ggcaagaatc ctggctgtgg aaagatacct aaaggatcaa cagctcctgg
     1921 ggatttgggg ttgctctgga aaactcattt gcaccactgc tgtgccttgg aatgctagtt
     1981 ggagtaataa atctctggaa cagatttgga atcacacgac ctggatggag tgggacagag
     2041 aaattaacaa ttacacaagc ttaatacact ccttaattga agaatcgcaa aaccagcaag
     2101 aaaagaatga acaagaatta ttggaattag ataaatgggc aagtttgtgg aattggttta
     2161 acataacaaa ttggctgtgg tatataaaat tattcataat gatagtagga ggcttggtag
     2221 gtttaagaat agtttttgct gtactttcta tagtgaatag agttaggcag ggatattcac
     2281 cattatcgtt tcagacccac ctcccaaccc cgaggggacc cgacaggccc gaaggaatag
     2341 aagaagaagg tggagagaga gacagagaca gatccattcg attagtgaac ggatcggcac
     2401 tgcgtgcgcc aattctgcag acaaatggca gtattcatcc acaattttaa aagaaaaggg
     2461 gggattgggg ggtacagtgc aggggaaaga atagtagaca taatagcaac agacatacaa
     2521 actaaagaat tacaaaaaca aattacaaaa attcaaaatt ttcgggttta ttacagggac
     2581 agcagagatc cagtttggtt agtaccgggc ccgctctaga atgagatcaa ggtcgcgaac
     2641 ctgaatgcag tctctcggag ggcgaagaat cctgcaggct cgagccaagg tcgggcagga
     2701 agagggccta tttcccatga ttccttcata tttgcatata cgatacaagg ctgttagaga
     2761 gataattgga attaatttga ctgtaaacac aaagatatta gtacaaaata cgtgacgtag
     2821 aaagtaataa tttcttgggt agtttgcagt tttaaaatta tgttttaaaa tggactatca
     2881 tatgcttacc gtaacttgaa agtatttcga tttcttggct ttatatatct tgtggaaagc
     2941 caccttgttg gagactcgcg tgtggcccca gtttcagagc taagcacaag agtgcatagc
     3001 aagttgaaat aaggctagtc cgtttacaac ttgaaaaagt ggcacccgag tcgggtgctt
     3061 tttttgcatg cgcggcgcgc cctaggatcc attaggcggc cgcgtggata accgtattac
     3121 cgccatgcat gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc cgagaagttg
     3181 gggggagggg tcggcaattg aaccggtgcc tagagaaggt ggcgcggggt aaactgggaa
     3241 agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc gtatataagt
     3301 gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac acaggtaagt
     3361 gccgtgtgtg gttcccgcgg gcctggcctc tttacgggtt atggcccttg cgtgccttga
     3421 attacttcca cctggctgca gtacgtgatt cttgatcccg agcttcgggt tggaagtggg
     3481 tgggagagtt cgaggccttg cgcttaagga gccccttcgc ctcgtgcttg agttgaggcc
     3541 tggcctgggc gctggggccg ccgcgtgcga atctggtggc accttcgcgc ctgtctcgct
     3601 gctttcgata agtctctagc catttaaaat ttttgatgac ctgctgcgac gctttttttc
     3661 tggcaagata gtcttgtaaa tgcgggccaa gatctgcaca ctggtatttc ggtttttggg
     3721 gccgcgggcg gcgacggggc ccgtgcgtcc cagcgcacat gttcggcgag gcggggcctg
     3781 cgagcgcggc caccgagaat cggacggggg tagtctcaag ctggccggcc tgctctggtg
     3841 cctggcctcg cgccgccgtg tatcgccccg ccctgggcgg caaggctggc ccggtcggca
     3901 ccagttgcgt gagcggaaag atggccgctt cccggccctg ctgcagggag ctcaaaatgg
     3961 aggacgcggc gctcgggaga gcgggcgggt gagtcaccca cacaaaggaa aagggccttt
     4021 ccgtcctcag ccgtcgcttc atgtgactcc acggagtacc gggcgccgtc caggcacctc
     4081 gattagttct cgcgcttttg gagtacgtcg tctttaggtt ggggggaggg gttttatgcg
     4141 atggagtttc cccacactga gtgggtggag actgaagtta ggccagcttg gcacttgatg
     4201 taattctcct tggaatttgc cctttttgag tttggatctt ggttcattct caagcctcag
     4261 acagtggttc aaagtttttt tcttccattt caggtgtcgt gagctagccc accatgaccg
     4321 agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt cccccgggcc gtacgcaccc
     4381 tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgacccg gaccgccaca
     4441 tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc gacatcggca
     4501 aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg gagagcgtcg
     4561 aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc ggttcccggc
     4621 tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag gagcccgcgt
     4681 ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg ggcagcgccg
     4741 tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc ctggagacct
     4801 ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc gccgacgtcg
     4861 aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc ggatcgggag
     4921 agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctggc ccaccggtcg
     4981 ccaccagcga gctgattaag gagaacatgc acatgaagct gtacatggag ggcaccgtgg
     5041 acaaccatca cttcaagtgc acatccgagg gcgaaggcaa gccctacgag ggcacccaga
     5101 ccatgagaat caaggtggtc gagggcggcc ctctcccctt cgccttcgac atcctggcta
     5161 ctagcttcct ctacggcagc aagaccttca tcaaccacac ccagggcatc cccgacttct
     5221 tcaagcagtc cttccctgag ggcttcacat gggagagagt caccacatac gaagacgggg
     5281 gcgtgctgac cgctacccag gacaccagcc tccaggacgg ctgcctcatc tacaacgtca
     5341 agatcagagg ggtgaacttc acatccaacg gccctgtgat gcagaagaaa acactcggct
     5401 gggaggcctt caccgagacg ctgtaccccg ctgacggcgg cctggaaggc agaaacgaca
     5461 tggccctgaa gctcgtgggc gggagccatc tgatcgcaaa catcaagacc acatatagat
     5521 ccaagaaacc cgctaagaac ctcaagatgc ctggcgtcta ctatgtggac tacagactgg
     5581 aaagaatcaa ggaggccaac aacgagacct acgtcgagca gcacgaggtg gcagtggcca
     5641 gatactgcga cctccctagc aaactggggc acaagcttaa ttaagaattc gtcgagggac
     5701 ctaataactt cgtatagcat acattatacg aagttataca tgtttaaggg ttccggttcc
     5761 actaggtaca attcgatatc aagcttatcg ataatcaacc tctggattac aaaatttgtg
     5821 aaagattgac tggtattctt aactatgttg ctccttttac gctatgtgga tacgctgctt
     5881 taatgccttt gtatcatgct attgcttccc gtatggcttt cattttctcc tccttgtata
     5941 aatcctggtt gctgtctctt tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg
     6001 tgtgcactgt gtttgctgac gcaaccccca ctggttgggg cattgccacc acctgtcagc
     6061 tcctttccgg gactttcgct ttccccctcc ctattgccac ggcggaactc atcgccgcct
     6121 gccttgcccg ctgctggaca ggggctcggc tgttgggcac tgacaattcc gtggtgttgt
     6181 cggggaaatc atcgtccttt ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg
     6241 ggacgtcctt ctgctacgtc ccttcggccc tcaatccagc ggaccttcct tcccgcggcc
     6301 tgctgccggc tctgcggcct cttccgcgtc ttcgccttcg ccctcagacg agtcggatct
     6361 ccctttgggc cgcctccccg catcgatacc gtcgacctcg atcgagacct agaaaaacat
     6421 ggagcaatca caagtagcaa tacagcagct accaatgctg attgtgcctg gctagaagca
     6481 caagaggagg aggaggtggg ttttccagtc acacctcagg tacctttaag accaatgact
     6541 tacaaggcag ctgtagatct tagccacttt ttaaaagaaa aggggggact ggaagggcta
     6601 attcactccc aacgaagaca agatatcctt gatctgtgga tctaccacac acaaggctac
     6661 ttccctgatt ggcagaacta cacaccaggg ccagggatca gatatccact gacctttgga
     6721 tggtgctaca agctagtacc agttgagcaa gagaaggtag aagaagccaa tgaaggagag
     6781 aacacccgct tgttacaccc tgtgagcctg catgggatgg atgacccgga gagagaagta
     6841 ttagagtgga ggtttgacag ccgcctagca tttcatcaca tggcccgaga gctgcatccg
     6901 gactgtactg ggtctctctg gttagaccag atctgagcct gggagctctc tggctaacta
     6961 gggaacccac tgcttaagcc tcaataaagc ttgccttgag tgcttcaagt agtgtgtgcc
     7021 cgtctgttgt gtgactctgg taactagaga tccctcagac ccttttagtc agtgtggaaa
     7081 atctctagca gcatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     7141 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     7201 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     7261 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     7321 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     7381 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     7441 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     7501 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     7561 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     7621 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     7681 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     7741 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     7801 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     7861 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     7921 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     7981 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     8041 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     8101 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     8161 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     8221 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     8281 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     8341 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     8401 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     8461 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     8521 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     8581 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     8641 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     8701 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     8761 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     8821 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     8881 tttccccgaa aagtgccacc tga
//
