LOCUS       sequence-340979-        4141 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPIE donor B5 (Zhong et al, eLife 2021), CDS of VCL exon21-mEGFP,
            translational phase (0-stop), excised by ACTB i4 sgRNA or DRS-1 
            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: pUC57-Amp_CRISPIE donor B5
FEATURES             Location/Qualifiers
     source          1..4141
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     165..184
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     424..445
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     regulatory      487..496
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(1055..1072)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1055..1071)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1064..1086)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1280..1299)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1399..1421
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1459..1477)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1545..1649
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1650..1718,1719..2510)
                     /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(1868..1887)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2681..3269
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3170..3189
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3423..3440
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3557..3578
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3593..3598,3599..3616,3617..3623)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3631..3647
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     3636..3658
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3655..3671
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3655..3671
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             complement(join(3912..4141,1..484,485..487,488..490))
                     /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(3957..3978)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 ccttgatgcc gttcttctgc ttgtcggcca tgatatagac gttgtggctg ttgtagttgt
       61 actccagctt gtgccccagg atgttgccgt cctccttgaa gtcgatgccc ttcagctcga
      121 tgcggttcac cagggtgtcg ccctcgaact tcacctcggc gcgggtcttg tagttgccgt
      181 cgtccttgaa gaagatggtg cgctcctgga cgtagccttc gggcatggcg gacttgaaga
      241 agtcgtgctg cttcatgtgg tcggggtagc ggctgaagca ctgcacgccg taggtcaggg
      301 tggtcacgag ggtgggccag ggcacgggca gcttgccggt ggtgcagatg aacttcaggg
      361 tcagcttgcc gtaggtggca tcgccctcgc cctcgccgga cacgctgaac ttgtggccgt
      421 ttacgtcgcc gtccagctcg accaggatgg gcaccacccc ggtgaacagc tcctcgccct
      481 tgctcaccat ggtggctgcg ctgcctccac cgctgccaga tctgagtccg gtaccaccag
      541 cactacctcc ggactggtac cagggagtct ttctaaccca gcgcagtgta aatccagcat
      601 ctgttcgaat tttgattgaa gcagcttcag cttcccgcac agtctccttc acagactgca
      661 tgaggttctg ggcattgtga accagcatct ctgtggcctg caaggaaaaa ggtactttcc
      721 gttacccagg gtccctgatg caggcctgtc tctaaaatag gtttcagcca ggagtgggat
      781 gcagctaacc ccactggtac ctggcatccc ccatatttgc ttgcatcact aggggttttc
      841 ccagatcacc aaaaagaacc caaaactggc catcgctgat tgtatactgt ggcttcagat
      901 agcttacagc atgtcctctt tcttggtctt caaacatgct tctccctcct gtaccgtact
      961 cggaagcgtc gcggccgcct gtaccgtact cggaagcgtc gaattcctcc gaccagtgtt
     1021 tgccttttac tagtatccca atacgcgtca attcactggc cgtcgtttta caacgtcgtg
     1081 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca
     1141 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga
     1201 atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc
     1261 gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagccccgac
     1321 acccgccaac acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca
     1381 gacaagctgt gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga
     1441 aacgcgcgag acgaaagggc ctcgtgatac gcctattttt ataggttaat gtcatgataa
     1501 taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga acccctattt
     1561 gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa ccctgataaa
     1621 tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt gtcgccctta
     1681 ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg ctggtgaaag
     1741 taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg gatctcaaca
     1801 gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg agcactttta
     1861 aagttctgct atgtggcgcg gtattatccc gtattgacgc cgggcaagag caactcggtc
     1921 gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca gaaaagcatc
     1981 ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg agtgataaca
     2041 ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc gcttttttgc
     2101 acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg aatgaagcca
     2161 taccaaacga cgagcgtgac accacgatgc ctgtagcaat ggcaacaacg ttgcgcaaac
     2221 tattaactgg cgaactactt actctagctt cccggcaaca attaatagac tggatggagg
     2281 cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg tttattgctg
     2341 ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg gggccagatg
     2401 gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact atggatgaac
     2461 gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa ctgtcagacc
     2521 aagtttactc atatatactt tagattgatt taaaacttca tttttaattt aaaaggatct
     2581 aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag ttttcgttcc
     2641 actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc
     2701 gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg
     2761 atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa
     2821 atactgttct tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc
     2881 ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt
     2941 gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa
     3001 cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc
     3061 tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc
     3121 cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct
     3181 ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat
     3241 gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc
     3301 tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg
     3361 ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc
     3421 gcagcgagtc agtgagcgag gaagcggaag agcgcccaat acgcaaaccg cctctccccg
     3481 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     3541 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     3601 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     3661 acagctatga ccatgattac gccaagctcg gcgcgccatt gggataagct tcctccgacc
     3721 agtgtttgcc ttttctgcag gacgcttccg agtacggtac agggatatcg acgcttccga
     3781 gtacggtaca ggtcctccac cctcacccca cctccactcc cctggggcat gcccctctac
     3841 taaggcaata cccttaatgc accccagaga ttcaggaaga aagaatcctt actgcggcag
     3901 gaccttccgg attacttgta cagctcgtcc atgccgagag tgatcccggc ggcggtcacg
     3961 aactccagca ggaccatgtg atcgcgcttc tcgttggggt ctttgctcag cttggactgg
     4021 gtgctcaggt agtggttgtc gggcagcagc acggggccgt cgccgatggg ggtgttctgc
     4081 tggtagtggt cggcgagctg cacgctgccg tcctcgatgt tgtggcggat cttgaagttc
     4141 a
//
