LOCUS       sequence-340938-        3852 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPIE donor B3 (Zhong et al, eLife 2021), mTurquoise2 
            translational phase (0-0), 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 3852)
  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 3852)
  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 B3
FEATURES             Location/Qualifiers
     source          1..3852
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(163..182)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     510..531
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(864..880)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(864..880)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(877..899)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    888..904
                     /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)."
     promoter        complement(join(912..918,919..936,937..942))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    957..978
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1095..1112)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1266..1854)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1346..1365)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2025..2816,2817..2885))
                     /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     2648..2667
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2886..2990)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     3058..3076
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(3114..3136)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     3236..3255
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3449..3471
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3463..3480
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3464..3480
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      3703..3712
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3709..3711,3712..3714,3715..3852,1..573)
                     /codon_start=1
                     /product="enhanced monomeric variant of CFP (Goedhart et 
                     al., 2012)"
                     /label=mTurquoise2
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSWGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYFSDNVYITADKQKNGIK
                     ANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3754..3775)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
ORIGIN
        1 tgcaccaccg gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gtcctggggc
       61 gtgcagtgct tcgcccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc
      121 atgcccgaag gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag
      181 acccgcgccg aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc
      241 atcgacttca aggaggacgg caacatcctg gggcacaagc tggagtacaa ctactttagc
      301 gacaacgtct atatcaccgc cgacaagcag aagaacggca tcaaggccaa cttcaagatc
      361 cgccacaaca tcgaggacgg cggcgtgcag ctcgccgacc actaccagca gaacaccccc
      421 atcggcgacg gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccaagctg
      481 agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc
      541 gggatcactc tcggcatgga cgagctgtac aagtccggag gtagtgctgg tggtaccgga
      601 ctcagatctg gcagcggtgg aggcagcgca ggtcctgccg cagtaaggat tctttcttcc
      661 tgaatctctg gggtgcatta agggtattgc cttagtagag gggcatgccc caggggagtg
      721 gaggtggggt gagggtggag gacctgtacc gtactcggaa gcgtcgatat ccctgtaccg
      781 tactcggaag cgtcctgcag aaaaggcaaa cactggtcgg aggaagctta tcccaatggc
      841 gcgccgagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
      901 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
      961 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     1021 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     1081 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     1141 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     1201 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     1261 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     1321 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     1381 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     1441 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     1501 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     1561 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     1621 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     1681 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
     1741 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     1801 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     1861 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     1921 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     1981 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     2041 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     2101 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     2161 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     2221 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     2281 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     2341 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     2401 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     2461 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     2521 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     2581 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     2641 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     2701 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     2761 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     2821 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     2881 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     2941 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     3001 cgaaaagtgc cacctgacgt ctaagaaacc attattatca tgacattaac ctataaaaat
     3061 aggcgtatca cgaggccctt tcgtctcgcg cgtttcggtg atgacggtga aaacctctga
     3121 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     3181 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gctggcttaa ctatgcggca
     3241 tcagagcaga ttgtactgag agtgcaccat atgcggtgtg aaataccgca cagatgcgta
     3301 aggagaaaat accgcatcag gcgccattcg ccattcaggc tgcgcaactg ttgggaaggg
     3361 cgatcggtgc gggcctcttc gctattacgc cagctggcga aagggggatg tgctgcaagg
     3421 cgattaagtt gggtaacgcc agggttttcc cagtcacgac gttgtaaaac gacggccagt
     3481 gaattgacgc gtattgggat actagtaaaa ggcaaacact ggtcggagga attcgacgct
     3541 tccgagtacg gtacaggcgg ccgcgacgct tccgagtacg gtacaggcaa attgctccag
     3601 aagggtgaga aggctagaga tgtgggagaa ctgggtggaa ggcagaatcg accacgtcta
     3661 aggttgtggg tttactgtgt cttacagttg aaggaaaccg gtgccaccat ggtgagcaag
     3721 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     3781 ggccacaagt tcagcgtgtc cggcgagggc gagggcgatg ccacctacgg caagctgacc
     3841 ctgaagttca tc
//
