LOCUS       sequence-341020-        4155 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DRS-2 sgRNA expression under a U6 promotor and CRISPIE donor B10 
            (Zhong et al, eLife 2021), mEGFP translational phase (2-stop), 
            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 4155)
  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 4155)
  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 B10
FEATURES             Location/Qualifiers
     source          1..4155
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     177..196
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     436..457
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     misc_RNA        complement(820..895)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(916..1164)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(974..993)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(1144..1164)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     rep_origin      complement(1226..1814)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1306..1325)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1985..2776,2777..2845))
                     /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     2608..2627
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2846..2950)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3018..3036
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3074..3096)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3196..3215
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3232..3687)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3369..3390)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3581..3600
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(join(3938..4155,1..496,497..499,500..502))
                     /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(3983..4004)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 tcttgaagtt caccttgatg ccgttcttct gcttgtcggc catgatatag acgttgtggc
       61 tgttgtagtt gtactccagc ttgtgcccca ggatgttgcc gtcctccttg aagtcgatgc
      121 ccttcagctc gatgcggttc accagggtgt cgccctcgaa cttcacctcg gcgcgggtct
      181 tgtagttgcc gtcgtccttg aagaagatgg tgcgctcctg gacgtagcct tcgggcatgg
      241 cggacttgaa gaagtcgtgc tgcttcatgt ggtcggggta gcggctgaag cactgcacgc
      301 cgtaggtcag ggtggtcacg agggtgggcc agggcacggg cagcttgccg gtggtgcaga
      361 tgaacttcag ggtcagcttg ccgtaggtgg catcgccctc gccctcgccg gacacgctga
      421 acttgtggcc gtttacgtcg ccgtccagct cgaccaggat gggcaccacc ccggtgaaca
      481 gctcctcgcc cttgctcacc attgcgctgc ctccaccgct gccagatctg agtccggtac
      541 caccagcact acctccggag gttccttcac tgtaagacac agtaaaccca caaccttaga
      601 cgtggtcgat tctgccttcc acccagttct cccacatctc tagccttctc acccttctgg
      661 agcaatttgc ctgtaccgta ctcggaagcg tcagcggccg ccccggctat gcttgccata
      721 cgcactagtt ctagagccat ttgtctgcag aattggcgca cgcgctaaaa acggactagc
      781 cttattttaa cttgctattt ctagctctaa aacaaaaaag caccgactcg gtgccacttt
      841 ttcaagttga taacggacta gccttatttt aacttgctat ttctagctct aaaaccggct
      901 atgcttgcca tacgcggtgt ttcgtccttt ccacaagata tataaagcca agaaatcgaa
      961 atactttcaa gttacggtaa gcatatgata gtccatttta aaacataatt ttaaaactgc
     1021 aaactaccca agaaattatt actttctacg tcacgtattt tgtactaata tctttgtgtt
     1081 tacagtcaaa ttaattccaa ttatctctct aacagccttg tatcgtatat gcaaatatga
     1141 aggaatcatg ggaaataggc cctcacatgt gagcaaaagg ccagcaaaag gccaggaacc
     1201 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
     1261 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
     1321 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
     1381 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     1441 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     1501 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     1561 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     1621 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     1681 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     1741 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     1801 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     1861 aaaactcacg ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc
     1921 ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
     1981 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
     2041 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg
     2101 gccccagtgc tgcaatgata ccgcggcttc cacgctcacc ggctccagat ttatcagcaa
     2161 taaaccagcc agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca
     2221 tccagtctat taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc
     2281 gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
     2341 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
     2401 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat
     2461 cactcatggt tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct
     2521 tttctgtgac tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga
     2581 gttgctcttg cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag
     2641 tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
     2701 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
     2761 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg
     2821 cgacacggaa atgttgaata ctcatactct tcctttttca atattattga agcatttatc
     2881 agggttattg tctcatgagc ggatacatat ttgaatgtat ttagaaaaat aaacaaatag
     2941 gggttccgcg cacatttccc cgaaaagtgc cacctgacgt ctaagaaacc attattatca
     3001 tgacattaac ctataaaaat aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg
     3061 atgacggtga aaacctctga cacatgcagc tcccggagac ggtcacagct tgtctgtaag
     3121 cggatgccgg gagcagacaa gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg
     3181 gctggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat aaaattgtaa
     3241 acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca ttttttaacc
     3301 aatagaccga aatcggcaaa atcccttata aatcaaaaga atagcccgag atagagttga
     3361 gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc aacgtcaaag
     3421 ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc aaatcaagtt
     3481 ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc ccccgattta
     3541 gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa gcgaaaggag
     3601 cgggcgctaa ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc acacccgccg
     3661 cgcttaatgc gccgctacag ggcgcgtact atggttgctt tgacgtatgc ggtgtgaaat
     3721 accgcacaga tgcgtaagga gaaaataccg catcaggcgc caagcttccc cggctatgct
     3781 tgccatacgc gatatcggat ccgacgcttc cgagtacggt acaggtcctc caccctcacc
     3841 ccacctccac tcccctgggg catgcccctc tactaaggca atacccttaa tgcaccccag
     3901 agattcagga agaaagaatc cttactgcgg caggacctta cttgtacagc tcgtccatgc
     3961 cgagagtgat cccggcggcg gtcacgaact ccagcaggac catgtgatcg cgcttctcgt
     4021 tggggtcttt gctcagcttg gactgggtgc tcaggtagtg gttgtcgggc agcagcacgg
     4081 ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc gagctgcacg ctgccgtcct
     4141 cgatgttgtg gcgga
//
