LOCUS       sequence-454346-        8841 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Lentiviral backbone used when cloning pooled prime editing guide RNA
            libraries already containing a tevopreQ1 RNA motif.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8841)
  AUTHORS   Cirincione A, Simpson D, Yan W, McNulty R, Ravisankar P, Solley SC, 
            Yan J, Lim F, Farley EK, Singh M, Adamson B
  TITLE     A benchmarked, high-efficiency prime editing platform for 
            multiplexed dropout screening.
  JOURNAL   Nat Methods. 2024 Nov 19. doi: 10.1038/s41592-024-02502-4.
  PUBMED    39562753
REFERENCE   2  (bases 1 to 8841)
  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: "Nat 
            Methods. 2024 Nov 19. doi: 10.1038/s41592-024-02502-4."
COMMENT     Sequence Label: pAC025
FEATURES             Location/Qualifiers
     source          1..8841
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(47..66)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        238..617
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        619..817
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     772..792
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     LTR             835..1015
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     misc_feature    1062..1187
                     /label=HIV-1 Psi
                     /note="packaging signal of human immunodeficiency virus 
                     type 1"
     misc_feature    1684..1917
                     /label=RRE
                     /note="The Rev response element (RRE) of HIV-1 allows for 
                     Rev-dependent mRNA export from the nucleus to the 
                     cytoplasm."
     CDS             2102..2146
                     /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    2444..2561
                     /label=cPPT/CTS
                     /note="central polypurine tract and central termination 
                     sequence of HIV-1"
     protein_bind    complement(2814..2847)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     2851..2871
                     /label=mU6-F
                     /note="Mouse U6 promoter, forward primer"
     promoter        3080..3291
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          3292..4230
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4187..4207
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4251..4847
                     /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(4251..4270)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4747..4767
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4857..4910
                     /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             4923..5621
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(5641..5674)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5730..6318
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5783..5803)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6201..6212)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6321..6337)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6322..6338)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6847..7027
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7089..7677)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7169..7188)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7848..8639,8640..8708))
                     /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     8471..8490
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8709..8813)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
       61 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
      121 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
      181 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatat acgcgttgac
      241 attgattatt gactagttat taatagtaat caattacggg gtcattagtt catagcccat
      301 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctga ccgcccaacg
      361 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgcca atagggactt
      421 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggca gtacatcaag
      481 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatgg cccgcctggc
      541 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatc tacgtattag
      601 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgt ggatagcggt
      661 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagt ttgttttggc
      721 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg
      781 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctg tactgggtct
      841 ctctggttag accagatctg agcctgggag ctctctggct aactagggaa cccactgctt
      901 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtct gttgtgtgac
      961 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctc tagcagtggc
     1021 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcga cgcaggactc
     1081 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagt acgccaaaaa
     1141 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagt attaagcggg
     1201 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaag aaaaaatata
     1261 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagtt aatcctggcc
     1321 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaacca tcccttcaga
     1381 caggatcaga agaacttaga tcattatata atacagtagc aaccctctat tgtgtgcatc
     1441 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaa gagcaaaaca
     1501 aaagtaagac caccgcacag caagcggccg gccgctgatc ttcagacctg gaggaggaga
     1561 tatgagggac aattggagaa gtgaattata taaatataaa gtagtaaaaa ttgaaccatt
     1621 aggagtagca cccaccaagg caaagagaag agtggtgcag agagaaaaaa gagcagtggg
     1681 aataggagct ttgttccttg ggttcttggg agcagcagga agcactatgg gcgcagcgtc
     1741 aatgacgctg acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaacaa
     1801 tttgctgagg gctattgagg cgcaacagca tctgttgcaa ctcacagtct ggggcatcaa
     1861 gcagctccag gcaagaatcc tggctgtgga aagataccta aaggatcaac agctcctggg
     1921 gatttggggt tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg
     1981 gagtaataaa tctctggaac agatttggaa tcacacgacc tggatggagt gggacagaga
     2041 aattaacaat tacacaagct taatacactc cttaattgaa gaatcgcaaa accagcaaga
     2101 aaagaatgaa caagaattat tggaattaga taaatgggca agtttgtgga attggtttaa
     2161 cataacaaat tggctgtggt atataaaatt attcataatg atagtaggag gcttggtagg
     2221 tttaagaata gtttttgctg tactttctat agtgaataga gttaggcagg gatattcacc
     2281 attatcgttt cagacccacc tcccaacccc gaggggaccc gacaggcccg aaggaataga
     2341 agaagaaggt ggagagagag acagagacag atccattcga ttagtgaacg gatcggcact
     2401 gcgtgcgcca attctgcaga caaatggcag tattcatcca caattttaaa agaaaagggg
     2461 ggattggggg gtacagtgca ggggaaagaa tagtagacat aatagcaaca gacatacaaa
     2521 ctaaagaatt acaaaaacaa attacaaaaa ttcaaaattt tcgggtttat tacagggaca
     2581 gcagagatcc agtttggtta gtaccgggcc cgctctagag atccgacgcg ccatctctag
     2641 gcccgcgccg gccccctcgc acggacttgt gggagaagct cggctactcc cctgccccgg
     2701 ttaatttgca tataatattt cctagtaact atagaggctt aatgtgcgat aaaagacaga
     2761 taatctgttc tttttaatac tagctacatt ttacatgata ggcttggatt tctataactt
     2821 cgtatagcat acattatacg aagttataaa cagcacaaaa ggaaactcac cctaactgta
     2881 aagtaattgt gtgttttgag actataagta tcccttggag aaccaccttg ttgggagacg
     2941 cctgcaggct gctaagctag gcgcgcccgt ctcatttttt tctcgagtac taggatccag
     3001 atcggaagag cacacgtctg aactccagtc acgcggccgc gtggataacc gtattaccgc
     3061 catgcatgtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
     3121 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
     3181 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
     3241 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
     3301 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
     3361 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
     3421 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
     3481 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
     3541 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
     3601 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
     3661 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
     3721 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
     3781 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
     3841 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
     3901 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
     3961 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
     4021 tagttctcgc gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     4081 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     4141 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     4201 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag ctagcccacc atgaccgagt
     4261 acaagcccac ggtgcgcctc gccacccgcg acgacgtccc ccgggccgta cgcaccctcg
     4321 ccgccgcgtt cgccgactac cccgccacgc gccacaccgt cgacccggac cgccacatcg
     4381 agcgggtcac cgagctgcaa gaactcttcc tcacgcgcgt cgggctcgac atcggcaagg
     4441 tgtgggtcgc ggacgacggc gccgcggtgg cggtctggac cacgccggag agcgtcgaag
     4501 cgggggcggt gttcgccgag atcggcccgc gcatggccga gttgagcggt tcccggctgg
     4561 ccgcgcagca acagatggaa ggcctcctgg cgccgcaccg gcccaaggag cccgcgtggt
     4621 tcctggccac cgtcggcgtg tcgcccgacc accagggcaa gggtctgggc agcgccgtcg
     4681 tgctccccgg agtggaggcg gccgagcgcg ccggggtgcc cgccttcctg gagacctccg
     4741 cgccccgcaa cctccccttc tacgagcggc tcggcttcac cgtcaccgcc gacgtcgagg
     4801 tgcccgaagg accgcgcacc tggtgcatga cccgcaagcc cggtgccgga tcgggagagg
     4861 gcagaggaag tctgctaaca tgcggtgacg tcgaggagaa tcctggccca ccggtcgcca
     4921 ccagcgagct gattaaggag aacatgcaca tgaagctgta catggagggc accgtggaca
     4981 accatcactt caagtgcaca tccgagggcg aaggcaagcc ctacgagggc acccagacca
     5041 tgagaatcaa ggtggtcgag ggcggccctc tccccttcgc cttcgacatc ctggctacta
     5101 gcttcctcta cggcagcaag accttcatca accacaccca gggcatcccc gacttcttca
     5161 agcagtcctt ccctgagggc ttcacatggg agagagtcac cacatacgaa gacgggggcg
     5221 tgctgaccgc tacccaggac accagcctcc aggacggctg cctcatctac aacgtcaaga
     5281 tcagaggggt gaacttcaca tccaacggcc ctgtgatgca gaagaaaaca ctcggctggg
     5341 aggccttcac cgagaccctg taccccgctg acggcggcct ggaaggcaga aacgacatgg
     5401 ccctgaagct cgtgggcggg agccatctga tcgcaaacat caagaccaca tatagatcca
     5461 agaaacccgc taagaacctc aagatgcctg gcgtctacta tgtggactac agactggaaa
     5521 gaatcaagga ggccaacaac gagacctacg tcgagcagca cgaggtggca gtggccagat
     5581 actgcgacct ccctagcaaa ctggggcaca agcttaatta agaattcgtc gagggaccta
     5641 ataacttcgt atagcataca ttatacgaag ttatacatgt ttaagggttc cggttccact
     5701 aggtacaatt cgatatcaag cttatcgata atcaacctct ggattacaaa atttgtgaaa
     5761 gattgactgg tattcttaac tatgttgctc cttttacgct atgtggatac gctgctttaa
     5821 tgcctttgta tcatgctatt gcttcccgta tggctttcat tttctcctcc ttgtataaat
     5881 cctggttgct gtctctttat gaggagttgt ggcccgttgt caggcaacgt ggcgtggtgt
     5941 gcactgtgtt tgctgacgca acccccactg gttggggcat tgccaccacc tgtcagctcc
     6001 tttccgggac tttcgctttc cccctcccta ttgccacggc ggaactcatc gccgcctgcc
     6061 ttgcccgctg ctggacaggg gctcggctgt tgggcactga caattccgtg gtgttgtcgg
     6121 ggaaatcatc gtcctttcct tggctgctcg cctgtgttgc cacctggatt ctgcgcggga
     6181 cgtccttctg ctacgtccct tcggccctca atccagcgga ccttccttcc cgcggcctgc
     6241 tgccggctct gcggcctctt ccgcgtcttc gccttcgccc tcagacgagt cggatctccc
     6301 tttgggccgc ctccccgcat cgataccgtc gacctcgatc gagacctaga aaaacatgga
     6361 gcaatcacaa gtagcaatac agcagctacc aatgctgatt gtgcctggct agaagcacaa
     6421 gaggaggagg aggtgggttt tccagtcaca cctcaggtac ctttaagacc aatgacttac
     6481 aaggcagctg tagatcttag ccacttttta aaagaaaagg ggggactgga agggctaatt
     6541 cactcccaac gaagacaaga tatccttgat ctgtggatct accacacaca aggctacttc
     6601 cctgattggc agaactacac accagggcca gggatcagat atccactgac ctttggatgg
     6661 tgctacaagc tagtaccagt tgagcaagag aaggtagaag aagccaatga aggagagaac
     6721 acccgcttgt tacaccctgt gagcctgcat gggatggatg acccggagag agaagtatta
     6781 gagtggaggt ttgacagccg cctagcattt catcacatgg cccgagagct gcatccggac
     6841 tgtactgggt ctctctggtt agaccagatc tgagcctggg agctctctgg ctaactaggg
     6901 aacccactgc ttaagcctca ataaagcttg ccttgagtgc ttcaagtagt gtgtgcccgt
     6961 ctgttgtgtg actctggtaa ctagagatcc ctcagaccct tttagtcagt gtggaaaatc
     7021 tctagcagca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     7081 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     7141 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     7201 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     7261 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     7321 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     7381 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     7441 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     7501 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     7561 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     7621 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     7681 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     7741 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     7801 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     7861 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     7921 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     7981 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     8041 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     8101 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     8161 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     8221 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     8281 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     8341 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     8401 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     8461 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     8521 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     8581 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     8641 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     8701 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     8761 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     8821 ccccgaaaag tgccacctga c
//
