LOCUS       sequence-341024-        4158 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DRS-2 sgRNA expression under a U6 promotor and CRISPIE donor B7 
            (Zhong et al, eLife 2021), mEGFP translational phase (0-0), 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 4158)
  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 4158)
  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 B7
FEATURES             Location/Qualifiers
     source          1..4158
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(218..237)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     565..586
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      931..1386
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1018..1037)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1228..1249
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1403..1422)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1522..1544
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1582..1600)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1668..1772
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1773..1841,1842..2633)
                     /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     complement(1991..2010)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2804..3392
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3293..3312
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3454..3702
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3454..3474
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3625..3644
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3723..3798
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     regulatory      4064..4073
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4070..4072,4073..4075,4076..4158,1..628)
                     /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(4115..4136)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
       61 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
      121 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
      181 cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
      241 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
      301 cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa
      361 cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca
      421 caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg
      481 cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtcca agctgagcaa
      541 agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat
      601 cactctcggc atggacgagc tgtacaagtc cggaggtagt gctggtggca ccggactcag
      661 atctggcagc ggtggaggca gcgcaggtcc tgccgcagta aggattcttt cttcctgaat
      721 ctctggggtg cattaagggt attgccttag tagaggggca tgccccaggg gagtggaggt
      781 ggggtgaggg tggaggacct gtaccgtact cggaagcgtc ggatcatccc cggctatgct
      841 tgccatacgc aagcttggcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt
      901 tcacaccgca tacgtcaaag caaccatagt acgcgccctg tagcggcgca ttaagcgcgg
      961 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcctta gcgcccgctc
     1021 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa
     1081 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac
     1141 ttgatttggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt
     1201 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca
     1261 actctatctc gggctattct tttgatttat aagggatttt gccgatttcg gtctattggt
     1321 taaaaaatga gctgatttaa caaaaattta acgcgaattt taacaaaata ttaacgttta
     1381 caattttatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagcccc
     1441 gacacccgcc aacacccgct gacgcgccct gacgggcttg tctgctcccg gcatccgctt
     1501 acagacaagc tgtgaccgtc tccgggagct gcatgtgtca gaggttttca ccgtcatcac
     1561 cgaaacgcgc gagacgaaag ggcctcgtga tacgcctatt tttataggtt aatgtcatga
     1621 taataatggt ttcttagacg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     1681 tttgtttatt tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat
     1741 aaatgcttca ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc
     1801 ttattccctt ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga
     1861 aagtaaaaga tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca
     1921 acagcggtaa gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt
     1981 ttaaagttct gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg
     2041 gtcgccgcat acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc
     2101 atcttacgga tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata
     2161 acactgcggc caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt
     2221 tgcacaacat gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag
     2281 ccataccaaa cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca
     2341 aactattaac tggcgaacta cttactctag cttcccggca acaattaata gactggatgg
     2401 aggcggataa agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg
     2461 ctgataaatc tggagccggt gagcgtggaa gccgcggtat cattgcagca ctggggccag
     2521 atggtaagcc ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg
     2581 aacgaaatag acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag
     2641 accaagttta ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga
     2701 tctaggtgaa gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt
     2761 tccactgagc gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc
     2821 tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc
     2881 cggatcaaga gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac
     2941 caaatactgt tcttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac
     3001 cgcctacata cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt
     3061 cgtgtcttac cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct
     3121 gaacgggggg ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat
     3181 acctacagcg tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt
     3241 atccggtaag cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg
     3301 cctggtatct ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt
     3361 gatgctcgtc aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt
     3421 tcctggcctt ttgctggcct tttgctcaca tgtgagggcc tatttcccat gattccttca
     3481 tatttgcata tacgatacaa ggctgttaga gagataattg gaattaattt gactgtaaac
     3541 acaaagatat tagtacaaaa tacgtgacgt agaaagtaat aatttcttgg gtagtttgca
     3601 gttttaaaat tatgttttaa aatggactat catatgctta ccgtaacttg aaagtatttc
     3661 gatttcttgg ctttatatat cttgtggaaa ggacgaaaca ccgcgtatgg caagcatagc
     3721 cggttttaga gctagaaata gcaagttaaa ataaggctag tccgttatca acttgaaaaa
     3781 gtggcaccga gtcggtgctt ttttgtttta gagctagaaa tagcaagtta aaataaggct
     3841 agtccgtttt tagcgcgtgc gccaattctg cagacaaatg gctctagaac tagtgcgtat
     3901 ggcaagcata gccggggcgg ccgctgacgc ttccgagtac ggtacaggca aattgctcca
     3961 gaagggtgag aaggctagag atgtgggaga actgggtgga aggcagaatc gaccacgtct
     4021 aaggttgtgg gtttactgtg tcttacagtt gaaggaaacc ggtgccacca tggtgagcaa
     4081 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     4141 cggccacaag ttcagcgt
//
