LOCUS       sequence-450494-        4923 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Constitutively expresses mCherry as a transfection marker. Expresses
            non-fluorescent EGFP, deactivated by Y93H mutation, that can be 
            reactivated by active cytosine base editing.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4923)
  AUTHORS   Osgood NRB, Zawalick NM, Sawyer CB, Cowan QT, Gu S, Mawson SJ, 
            Ranzau BL, Li L, Gymrek M, Goren A, Komor AC
  TITLE     Genome editing with programmable base editors in human cells
  JOURNAL   Methods in Enzymology, February 5, 2025
REFERENCE   2  (bases 1 to 4923)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Methods in 
            Enzymology, February 5, 2025"
COMMENT     Sequence Label: pCBE-dGFP
FEATURES             Location/Qualifiers
     source          1..4923
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(90..108)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     358..377
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             659..715
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             join(716..718,719..721,722..1432)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGHVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(761..782)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1022..1041)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1369..1390
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1445..1465
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1474..1491
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1514..1531)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1520..1744
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1815..1831)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1815..1831)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1828..1850)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1839..1855
                     /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(1863..1869,1870..1887,1888..1893))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1908..1929
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2046..2063)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2217..2805)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2297..2316)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2976..3767,3768..3836))
                     /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     3599..3618
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3837..3941)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(4020..4039)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        4211..4590
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        4591..4794
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     4745..4765
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     4836..4855
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        4836..4854
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      4862..4871
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(4868..4923,1..652)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
ORIGIN
        1 caaggtgcac atggagggct ccgtgaacgg ccacgagttc gagatcgagg gcgagggcga
       61 gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg gtggccccct
      121 gcccttcgcc tgggacatcc tgtcccctca gttcatgtac ggctccaagg cctacgtgaa
      181 gcaccccgcc gacatccccg actacttgaa gctgtccttc cccgagggct tcaagtggga
      241 gcgcgtgatg aacttcgagg acggcggcgt ggtgaccgtg acccaggact cctccctgca
      301 ggacggcgag ttcatctaca aggtgaagct gcgcggcacc aacttcccct ccgacggccc
      361 cgtaatgcag aagaagacca tgggctggga ggcctcctcc gagcggatgt accccgagga
      421 cggcgccctg aagggcgaga tcaagcagag gctgaagctg aaggacggcg gccactacga
      481 cgctgaggtc aagaccacct acaaggccaa gaagcccgtg cagctgcccg gcgcctacaa
      541 cgtcaacatc aagttggaca tcacctccca caacgaggac tacaccatcg tggaacagta
      601 cgaacgcgcc gagggccgcc actccaccgg cggcatggac gagctgtaca agagcggagc
      661 tactaacttc agcctgctga agcaggctgg agacgtggag gagaaccctg gacctatggt
      721 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
      781 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
      841 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
      901 gaccaccctg acctatggag tgcagtgctt cagccgctac cccgaccaca tgaagcagca
      961 cgacttcttc aagtccgcca tgcccgaagg tcacgtacag gagcgcacga tcttcttcaa
     1021 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1081 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1141 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     1201 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     1261 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1321 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1381 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtctggtgg
     1441 ttctcccaag aagaaaagga aagtctaacc ggtcatcatc accatcacca ttgagtttaa
     1501 acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc
     1561 cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag
     1621 gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag
     1681 gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct
     1741 atggcttctg aggcggaaag aaccagctgg ggctcgatac cgtcgacctc tagctagagc
     1801 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     1861 cacaacatac gagccggaag cataaagtgt aaagcctagg atgcctaatg agtgagctaa
     1921 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     1981 ctgcattaat gaatcggcca acgcgcggga agaggcggtt tgcgtattgg gcgctcttcc
     2041 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     2101 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     2161 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     2221 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     2281 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     2341 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     2401 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     2461 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     2521 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     2581 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     2641 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     2701 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     2761 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     2821 ttttctacgg ggtctgacac tcagtggaac gaaaactcac gttaagggat tttggtcatg
     2881 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     2941 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     3001 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     3061 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     3121 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     3181 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     3241 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     3301 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     3361 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     3421 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     3481 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     3541 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     3601 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     3661 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     3721 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     3781 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     3841 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     3901 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     3961 ccacctgacg tcgacggatc gggagatcga tctcccgatc ccctagggtc gactctcagt
     4021 acaatctgct ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag
     4081 gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat
     4141 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
     4201 tatacgcgtt gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta
     4261 gttcatagcc catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc
     4321 tgaccgccca acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg
     4381 ccaataggga ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg
     4441 gcagtacatc aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa
     4501 tggcccgcct ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac
     4561 atctacgtat tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg
     4621 cgtggatagc ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg
     4681 agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca
     4741 ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctggttta
     4801 gtgaaccgtc agatccgcta gagatccgcg gccgctaata cgactcacta tagggagagc
     4861 cgccaccatg gtgagcaagg gcgaggagga taacatggcc atcatcaagg agttcatgcg
     4921 ctt
//
