LOCUS       sequence-341030-        3858 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  DRS-2 sgRNA expression under a U6 promotor and HITI donor B6 (Zhong 
            et al, eLife 2021), mEGFP translational phase (1-1), excised by 
            DRS-2 sgRNA (with SpCas9)..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3858)
  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 3858)
  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 _HITI donor B6
FEATURES             Location/Qualifiers
     source          1..3858
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     185..204
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     444..465
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      507..516
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     misc_RNA        complement(644..719)
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     promoter        complement(740..988)
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     complement(798..817)
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     primer_bind     complement(968..988)
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     rep_origin      complement(1050..1638)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1130..1149)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1809..2600,2601..2669))
                     /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     2432..2451
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2670..2774)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2842..2860
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2898..2920)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3020..3039
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     rep_origin      complement(3056..3511)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3193..3214)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3405..3424
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     CDS             complement(join(3652..3858,1..504,505..507,508..510))
                     /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(3694..3715)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 gtggcggatc ttgaagttca ccttgatgcc gttcttctgc ttgtcggcca tgatatagac
       61 gttgtggctg ttgtagttgt actccagctt gtgccccagg atgttgccgt cctccttgaa
      121 gtcgatgccc ttcagctcga tgcggttcac cagggtgtcg ccctcgaact tcacctcggc
      181 gcgggtcttg tagttgccgt cgtccttgaa gaagatggtg cgctcctgga cgtagccttc
      241 gggcatggcg gacttgaaga agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca
      301 ctgcacgccg taggtcaggg tggtcacgag ggtgggccag ggcacgggca gcttgccggt
      361 ggtgcagatg aacttcaggg tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga
      421 cacgctgaac ttgtggccgt ttacgtcgcc gtccagctcg accaggatgg gcaccacccc
      481 ggtgaacagc tcctcgccct tgctcaccat ggtggcaccg gtagccccgg ctatgcttgc
      541 catacgcact agttctagag ccatttgtct gcagaattgg cgcacgcgct aaaaacggac
      601 tagccttatt ttaacttgct atttctagct ctaaaacaaa aaagcaccga ctcggtgcca
      661 ctttttcaag ttgataacgg actagcctta ttttaacttg ctatttctag ctctaaaacc
      721 ggctatgctt gccatacgcg gtgtttcgtc ctttccacaa gatatataaa gccaagaaat
      781 cgaaatactt tcaagttacg gtaagcatat gatagtccat tttaaaacat aattttaaaa
      841 ctgcaaacta cccaagaaat tattactttc tacgtcacgt attttgtact aatatctttg
      901 tgtttacagt caaattaatt ccaattatct ctctaacagc cttgtatcgt atatgcaaat
      961 atgaaggaat catgggaaat aggccctcac atgtgagcaa aaggccagca aaaggccagg
     1021 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     1081 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     1141 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     1201 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     1261 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     1321 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     1381 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     1441 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     1501 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     1561 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     1621 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     1681 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     1741 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     1801 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     1861 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     1921 tctggcccca gtgctgcaat gataccgcgg cttccacgct caccggctcc agatttatca
     1981 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     2041 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     2101 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     2161 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     2221 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     2281 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     2341 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     2401 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     2461 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     2521 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     2581 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     2641 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     2701 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     2761 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt
     2821 atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc
     2881 ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg
     2941 taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt
     3001 cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccataaaatt
     3061 gtaaacgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt
     3121 aaccaataga ccgaaatcgg caaaatccct tataaatcaa aagaatagcc cgagatagag
     3181 ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc
     3241 aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc acccaaatca
     3301 agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga
     3361 tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa
     3421 ggagcgggcg ctaaggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc
     3481 gccgcgctta atgcgccgct acagggcgcg tactatggtt gctttgacgt atgcggtgtg
     3541 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgccaagct tgcgtatggc
     3601 aagcatagcc ggggatgcgc tgcctccacc gctgccagat ctgagtccgg acttgtacag
     3661 ctcgtccatg ccgagagtga tcccggcggc ggtcacgaac tccagcagga ccatgtgatc
     3721 gcgcttctcg ttggggtctt tgctcagctt ggactgggtg ctcaggtagt ggttgtcggg
     3781 cagcagcacg gggccgtcgc cgatgggggt gttctgctgg tagtggtcgg cgagctgcac
     3841 gctgccgtcc tcgatgtt
//
