LOCUS       sequence-340928-        4141 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DRS-2 sgRNA expression under a U6 promotor and CRISPIE donor B8 
            (Zhong et al, eLife 2021), mEGFP translational phase (1-1), excised 
            by DRS-1 or DRS-2 sgRNA (with SpCas9)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4141)
  AUTHORS   Zhong H, Ceballos CC, Massengill CI, Muniak MA, Ma L, Qin M, Petrie 
            SK, Mao T
  TITLE     High-fidelity, efficient, and reversible labeling of endogenous 
            proteins using CRISPR-based designer exon insertion.
  JOURNAL   Elife. 2021 Jun 8;10. pii: 64911. doi: 10.7554/eLife.64911.
  PUBMED    34100715
REFERENCE   2  (bases 1 to 4141)
  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: "2021-06-8"; volume: "10. pii: 64911. doi"; pages: " 
            10.7554/eLife.64911"
COMMENT     Sequence Label: pX330 _CRISPIE donor B8
FEATURES             Location/Qualifiers
     source          1..4141
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     83..101
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(139..161)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     261..280
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(297..752)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(434..455)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     646..665
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(join(1040..1750,1751..1753,1754..1756))
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1082..1103)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     1431..1450
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1690..1711
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      1753..1762
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        complement(2026..2101)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(2122..2370)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(2180..2199)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(2350..2370)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     rep_origin      complement(2432..3020)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2512..2531)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3191..3982,3983..4051))
                     /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     3814..3833
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(4052..4141,1..15))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
       61 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc tcgcgcgttt
      121 cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca cagcttgtct
      181 gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg
      241 tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accataaaat
      301 tgtaaacgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
      361 taaccaatag accgaaatcg gcaaaatccc ttataaatca aaagaatagc ccgagataga
      421 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
      481 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat cacccaaatc
      541 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
      601 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
      661 aggagcgggc gctaaggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
      721 cgccgcgctt aatgcgccgc tacagggcgc gtactatggt tgctttgacg tatgcggtgt
      781 gaaataccgc acagatgcgt aaggagaaaa taccgcatca ggcgccaagc ttccccggct
      841 atgcttgcca tacgcgatat cgacgcttcc gagtacggta caggcaagac ccctcttccc
      901 acaccggcca cacccagtag agcagctcca tcaaatctca gcacaccctg tcctcggggc
      961 tcacgggact gcctggcctc tcacctgaga agcactcaga acctgcgctg cctccaccgc
     1021 tgccagatct gagtccggac ttgtacagct cgtccatgcc gagagtgatc ccggcggcgg
     1081 tcacgaactc cagcaggacc atgtgatcgc gcttctcgtt ggggtctttg ctcagcttgg
     1141 actgggtgct caggtagtgg ttgtcgggca gcagcacggg gccgtcgccg atgggggtgt
     1201 tctgctggta gtggtcggcg agctgcacgc tgccgtcctc gatgttgtgg cggatcttga
     1261 agttcacctt gatgccgttc ttctgcttgt cggccatgat atagacgttg tggctgttgt
     1321 agttgtactc cagcttgtgc cccaggatgt tgccgtcctc cttgaagtcg atgcccttca
     1381 gctcgatgcg gttcaccagg gtgtcgccct cgaacttcac ctcggcgcgg gtcttgtagt
     1441 tgccgtcgtc cttgaagaag atggtgcgct cctggacgta gccttcgggc atggcggact
     1501 tgaagaagtc gtgctgcttc atgtggtcgg ggtagcggct gaagcactgc acgccgtagg
     1561 tcagggtggt cacgagggtg ggccagggca cgggcagctt gccggtggtg cagatgaact
     1621 tcagggtcag cttgccgtag gtggcatcgc cctcgccctc gccggacacg ctgaacttgt
     1681 ggccgtttac gtcgccgtcc agctcgacca ggatgggcac caccccggtg aacagctcct
     1741 cgcccttgct caccatggtg gcaccggttt ccttcactgt aagacacagt aaacccacaa
     1801 ccttagacgt ggtcgattct gccttccacc cagttctccc acatctctag ccttctcacc
     1861 cttctggagc aatttgcctg taccgtactc ggaagcgtcg cggccgcccc ggctatgctt
     1921 gccatacgca ctagttctag agccatttgt ctgcagaatt ggcgcacgcg ctaaaaacgg
     1981 actagcctta ttttaacttg ctatttctag ctctaaaaca aaaaagcacc gactcggtgc
     2041 cactttttca agttgataac ggactagcct tattttaact tgctatttct agctctaaaa
     2101 ccggctatgc ttgccatacg cggtgtttcg tcctttccac aagatatata aagccaagaa
     2161 atcgaaatac tttcaagtta cggtaagcat atgatagtcc attttaaaac ataattttaa
     2221 aactgcaaac tacccaagaa attattactt tctacgtcac gtattttgta ctaatatctt
     2281 tgtgtttaca gtcaaattaa ttccaattat ctctctaaca gccttgtatc gtatatgcaa
     2341 atatgaagga atcatgggaa ataggccctc acatgtgagc aaaaggccag caaaaggcca
     2401 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     2461 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     2521 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     2581 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     2641 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     2701 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     2761 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     2821 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     2881 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     2941 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     3001 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     3061 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     3121 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     3181 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     3241 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     3301 catctggccc cagtgctgca atgataccgc ggcttccacg ctcaccggct ccagatttat
     3361 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     3421 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     3481 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     3541 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     3601 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     3661 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     3721 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     3781 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     3841 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     3901 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     3961 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     4021 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     4081 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     4141 a
//
