LOCUS       sequence-341005-        4187 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DRS-2 sgRNA expression under a U6 promotor and CRISPIE donor B9 
            (Zhong et al, eLife 2021), mEGFP translational phase (2-2), 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 4187)
  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 4187)
  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 B9
FEATURES             Location/Qualifiers
     source          1..4187
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(189..208)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     536..557
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     rep_origin      885..1340
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(972..991)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1182..1203
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(1357..1376)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1476..1498
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1536..1554)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1622..1726
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1727..1795,1796..2587)
                     /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(1945..1964)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2758..3346
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3247..3266
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        3408..3656
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3408..3428
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3579..3598
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3677..3752
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     CDS             join(4070..4072,4073..4075,4076..4187,1..599)
                     /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 acggcaagct gaccctgaag ttcatctgca ccaccggcaa gctgcccgtg ccctggccca
       61 ccctcgtgac caccctgacc tacggcgtgc agtgcttcag ccgctacccc gaccacatga
      121 agcagcacga cttcttcaag tccgccatgc ccgaaggcta cgtccaggag cgcaccatct
      181 tcttcaagga cgacggcaac tacaagaccc gcgccgaggt gaagttcgag ggcgacaccc
      241 tggtgaaccg catcgagctg aagggcatcg acttcaagga ggacggcaac atcctggggc
      301 acaagctgga gtacaactac aacagccaca acgtctatat catggccgac aagcagaaga
      361 acggcatcaa ggtgaacttc aagatccgcc acaacatcga ggacggcagc gtgcagctcg
      421 ccgaccacta ccagcagaac acccccatcg gcgacggccc cgtgctgctg cccgacaacc
      481 actacctgag cacccagtcc aagctgagca aagaccccaa cgagaagcgc gatcacatgg
      541 tcctgctgga gttcgtgacc gccgccggga tcactctcgg catggacgag ctgtacaagt
      601 ccggtctcag atccggcagc ggtggaggca gcgcggtcct gccgcagtaa ggattctttc
      661 ttcctgaatc tctggggtgc attaagggta ttgccttagt agaggggcat gccccagggg
      721 agtggaggtg gggtgagggt ggaggacctg taccgtactc ggaagcgtcg gatccgatat
      781 cgcgtatggc aagcatagcc ggggaagctt ggcgcctgat gcggtatttt ctccttacgc
      841 atctgtgcgg tatttcacac cgcatacgtc aaagcaacca tagtacgcgc cctgtagcgg
      901 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc
      961 cttagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc
     1021 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct
     1081 cgaccccaaa aaacttgatt tgggtgatgg ttcacgtagt gggccatcgc cctgatagac
     1141 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac
     1201 tggaacaaca ctcaactcta tctcgggcta ttcttttgat ttataaggga ttttgccgat
     1261 ttcggtctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa
     1321 aatattaacg tttacaattt tatggtgcac tctcagtaca atctgctctg atgccgcata
     1381 gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg cttgtctgct
     1441 cccggcatcc gcttacagac aagctgtgac cgtctccggg agctgcatgt gtcagaggtt
     1501 ttcaccgtca tcaccgaaac gcgcgagacg aaagggcctc gtgatacgcc tatttttata
     1561 ggttaatgtc atgataataa tggtttctta gacgtcaggt ggcacttttc ggggaaatgt
     1621 gcgcggaacc cctatttgtt tatttttcta aatacattca aatatgtatc cgctcatgag
     1681 acaataaccc tgataaatgc ttcaataata ttgaaaaagg aagagtatga gtattcaaca
     1741 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     1801 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     1861 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     1921 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     1981 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     2041 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     2101 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     2161 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     2221 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     2281 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     2341 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     2401 tggctggttt attgctgata aatctggagc cggtgagcgt ggaagccgcg gtatcattgc
     2461 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     2521 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     2581 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     2641 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta
     2701 acgtgagttt tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg
     2761 agatcctttt tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc
     2821 ggtggtttgt ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag
     2881 cagagcgcag ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa
     2941 gaactctgta gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc
     3001 cagtggcgat aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc
     3061 gcagcggtcg ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta
     3121 caccgaactg agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag
     3181 aaaggcggac aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct
     3241 tccaggggga aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga
     3301 gcgtcgattt ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc
     3361 ggccttttta cggttcctgg ccttttgctg gccttttgct cacatgtgag ggcctatttc
     3421 ccatgattcc ttcatatttg catatacgat acaaggctgt tagagagata attggaatta
     3481 atttgactgt aaacacaaag atattagtac aaaatacgtg acgtagaaag taataatttc
     3541 ttgggtagtt tgcagtttta aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa
     3601 cttgaaagta tttcgatttc ttggctttat atatcttgtg gaaaggacga aacaccgcgt
     3661 atggcaagca tagccggttt tagagctaga aatagcaagt taaaataagg ctagtccgtt
     3721 atcaacttga aaaagtggca ccgagtcggt gcttttttgt tttagagcta gaaatagcaa
     3781 gttaaaataa ggctagtccg tttttagcgc gtgcgccaat tctgcagaca aatggctcta
     3841 gaactagtgc gtatggcaag catagccggg gcggccgctg acgcttccga gtacggtaca
     3901 ggcaaattgc tccagaaggg tgagaaggct agagatgtgg gagaactggg tggaaggcag
     3961 aatcgaccac gtctaaggtt gtgggtttac tgtgtcttac agtgaaggaa cctccggagg
     4021 tagtgctggt ggtaccggac tcagatctgg cagcggtgga ggcagcgcaa tggtgagcaa
     4081 gggcgaggag ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa
     4141 cggccacaag ttcagcgtgt ccggcgaggg cgagggcgat gccacct
//
