LOCUS       sequence-374854-        8850 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  sgRNA targeting enhancer 4 of MYC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8850)
  AUTHORS   Lin X, Liu Y, Liu S, Zhu X, Wu L, Zhu Y, Zhao D, Xu X, Chemparathy 
            A, Wang H, Cao Y, Nakamura M, Noordermeer JN, La Russa M, Wong WH, 
            Zhao K, Qi LS
  TITLE     Nested epistasis enhancer networks for robust genome regulation.
  JOURNAL   Science. 2022 Aug 11:eabk3512. doi: 10.1126/science.abk3512.
  PUBMED    35951677
REFERENCE   2  (bases 1 to 8850)
  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: "Science. 
            2022 Aug 11:eabk3512. doi: 10.1126/science.abk3512."
COMMENT     Sequence Label: pSLQ5004_hU6_sgRNA targeting e4
FEATURES             Location/Qualifiers
     source          1..8850
                     /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"
     primer_bind     2646..2666
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     2817..2836
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        2915..2990
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        3089..3300
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     intron          3301..4239
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     4196..4216
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             4260..4856
                     /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(4260..4279)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4756..4776
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             4866..4919
                     /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             4932..5630
                     /codon_start=1
                     /product="monomeric blue fluorescent protein"
                     /label=TagBFP
                     /note="mammalian codon-optimized"
                     /translation="SELIKENMHMKLYMEGTVDNHHFKCTSEGEGKPYEGTQTMRIKVV
                     EGGPLPFAFDILATSFLYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTAT
                     QDTSLQDGCLIYNVKIRGVNFTSNGPVMQKKTLGWEAFTETLYPADGGLEGRNDMALKL
                     VGGSHLIANIKTTYRSKKPAKNLKMPGVYYVDYRLERIKEANNETYVEQHEVAVARYCD
                     LPSKLGHKLN"
     protein_bind    complement(5650..5683)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    5739..6327
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(5792..5812)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(6210..6221)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(6330..6346)
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6331..6347)
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     LTR             6856..7036
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from HIV-1"
     rep_origin      complement(7098..7686)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7178..7197)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7857..8648,8649..8717))
                     /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     8480..8499
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8718..8822)
                     /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 ccttttgctc attaattaag
     2641 gtaccgaggg cctatttccc atgattcctt catatttgca tatacgatac aaggctgtta
     2701 gagagataat tagaattaat ttgactgtaa acacaaagat attagtacaa aatacgtgac
     2761 gtagaaagta ataatttctt gggtagtttg cagttttaaa attatgtttt aaaatggact
     2821 atcatatgct taccgtaact tgaaagtatt tcgatttctt ggctttatat atcttgtgga
     2881 aagccagaaa catgggctgg ctgggtctgg tagtgtttta gagctagaaa tagcaagtta
     2941 aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc tttttttgaa
     3001 ttcgctagcc tagaggtacc tcgagtacta ggatccatta ggcggccgcg tggataaccg
     3061 tattaccgcc atgcatgtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag
     3121 aagttggggg gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac
     3181 tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat
     3241 ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag
     3301 gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg
     3361 ccttgaatta cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga
     3421 agtgggtggg agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt
     3481 gaggcctggc ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt
     3541 ctcgctgctt tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt
     3601 tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt
     3661 tttggggccg cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg
     3721 ggcctgcgag cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct
     3781 ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg
     3841 tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca
     3901 aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg
     3961 gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg
     4021 cacctcgatt agttctcgcg cttttggagt acgtcgtctt taggttgggg ggaggggttt
     4081 tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac
     4141 ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag
     4201 cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgagc tagcccacca
     4261 tgaccgagta caagcccacg gtgcgcctcg ccacccgcga cgacgtcccc cgggccgtac
     4321 gcaccctcgc cgccgcgttc gccgactacc ccgccacgcg ccacaccgtc gacccggacc
     4381 gccacatcga gcgggtcacc gagctgcaag aactcttcct cacgcgcgtc gggctcgaca
     4441 tcggcaaggt gtgggtcgcg gacgacggcg ccgcggtggc ggtctggacc acgccggaga
     4501 gcgtcgaagc gggggcggtg ttcgccgaga tcggcccgcg catggccgag ttgagcggtt
     4561 cccggctggc cgcgcagcaa cagatggaag gcctcctggc gccgcaccgg cccaaggagc
     4621 ccgcgtggtt cctggccacc gtcggcgtct cgcccgacca ccagggcaag ggtctgggca
     4681 gcgccgtcgt gctccccgga gtggaggcgg ccgagcgcgc cggggtgccc gccttcctgg
     4741 agacctccgc gccccgcaac ctccccttct acgagcggct cggcttcacc gtcaccgccg
     4801 acgtcgaggt gcccgaagga ccgcgcacct ggtgcatgac ccgcaagccc ggtgccggat
     4861 cgggagaggg cagaggaagt ctgctaacat gcggtgacgt cgaggagaat cctggcccac
     4921 cggtcgccac cagcgagctg attaaggaga acatgcacat gaagctgtac atggagggca
     4981 ccgtggacaa ccatcacttc aagtgcacat ccgagggcga aggcaagccc tacgagggca
     5041 cccagaccat gagaatcaag gtggtcgagg gcggccctct ccccttcgcc ttcgacatcc
     5101 tggctactag cttcctctac ggcagcaaga ccttcatcaa ccacacccag ggcatccccg
     5161 acttcttcaa gcagtccttc cctgagggct tcacatggga gagagtcacc acatacgaag
     5221 acgggggcgt gctgaccgct acccaggaca ccagcctcca ggacggctgc ctcatctaca
     5281 acgtcaagat cagaggggtg aacttcacat ccaacggccc tgtgatgcag aagaaaacac
     5341 tcggctggga ggccttcacc gagacgctgt accccgctga cggcggcctg gaaggcagaa
     5401 acgacatggc cctgaagctc gtgggcggga gccatctgat cgcaaacatc aagaccacat
     5461 atagatccaa gaaacccgct aagaacctca agatgcctgg cgtctactat gtggactaca
     5521 gactggaaag aatcaaggag gccaacaacg agacctacgt cgagcagcac gaggtggcag
     5581 tggccagata ctgcgacctc cctagcaaac tggggcacaa gcttaattaa gaattcgtcg
     5641 agggacctaa taacttcgta tagcatacat tatacgaagt tatacatgtt taagggttcc
     5701 ggttccacta ggtacaattc gatatcaagc ttatcgataa tcaacctctg gattacaaaa
     5761 tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta tgtggatacg
     5821 ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt ttctcctcct
     5881 tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc aggcaacgtg
     5941 gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt gccaccacct
     6001 gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg gaactcatcg
     6061 ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac aattccgtgg
     6121 tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc acctggattc
     6181 tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac cttccttccc
     6241 gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct cagacgagtc
     6301 ggatctccct ttgggccgcc tccccgcatc gataccgtcg acctcgatcg agacctagaa
     6361 aaacatggag caatcacaag tagcaataca gcagctacca atgctgattg tgcctggcta
     6421 gaagcacaag aggaggagga ggtgggtttt ccagtcacac ctcaggtacc tttaagacca
     6481 atgacttaca aggcagctgt agatcttagc cactttttaa aagaaaaggg gggactggaa
     6541 gggctaattc actcccaacg aagacaagat atccttgatc tgtggatcta ccacacacaa
     6601 ggctacttcc ctgattggca gaactacaca ccagggccag ggatcagata tccactgacc
     6661 tttggatggt gctacaagct agtaccagtt gagcaagaga aggtagaaga agccaatgaa
     6721 ggagagaaca cccgcttgtt acaccctgtg agcctgcatg ggatggatga cccggagaga
     6781 gaagtattag agtggaggtt tgacagccgc ctagcatttc atcacatggc ccgagagctg
     6841 catccggact gtactgggtc tctctggtta gaccagatct gagcctggga gctctctggc
     6901 taactaggga acccactgct taagcctcaa taaagcttgc cttgagtgct tcaagtagtg
     6961 tgtgcccgtc tgttgtgtga ctctggtaac tagagatccc tcagaccctt ttagtcagtg
     7021 tggaaaatct ctagcagcat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     7081 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     7141 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     7201 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     7261 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     7321 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     7381 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     7441 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     7501 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     7561 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     7621 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     7681 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     7741 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     7801 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     7861 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     7921 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     7981 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     8041 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     8101 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     8161 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     8221 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     8281 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     8341 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     8401 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     8461 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     8521 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     8581 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     8641 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     8701 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     8761 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     8821 cgcacatttc cccgaaaagt gccacctgac
//
