LOCUS       sequence-319108-        5849 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Transient Reporter for Editing Enrichment (TREE) Plasmid. GFP turns 
            on with adenosine base editing (ABE). 2 stop codons..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5849)
  AUTHORS   Brookhouser N, Nguyen T, Tekel SJ, Standage-Beier K, Wang X, Brafman
            DA
  TITLE     A Cas9-mediated adenosine transient reporter enables enrichment of 
            ABE-targeted cells.
  JOURNAL   BMC Biol. 2020 Dec 14;18(1):193. doi: 10.1186/s12915-020-00929-7.
  PUBMED    33317513
REFERENCE   2  (bases 1 to 5849)
  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: "BMC Biol.";
            date: "2020-12-14"; volume: "18(1):193. doi"; pages: " 
            10.1186/s12915-020-00929-7"
COMMENT     Sequence Label: pEF-XMAS-2xStop
FEATURES             Location/Qualifiers
     source          1..5849
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..1217
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          266..1208
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1165..1185
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1263..1272
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1269..1976
                     /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"
     primer_bind     complement(1414..1432)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     1682..1701
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             1983..2036
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(2070..2072,2073..2786)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(2070..2072,2073..2783)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(2112..2133)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2373..2392)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2720..2741
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2873..2892)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    2938..2993
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(2992..3011)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3352..3368)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3352..3368)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3365..3387)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3376..3392
                     /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(3400..3406,3407..3424,3425..3430))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3445..3466
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3525..3720
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3571..3706
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3726..3860
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3763..3782)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3817..3836
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3928..3945)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4099..4687)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4179..4198)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4858..5649,5650..5718))
                     /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     5481..5500
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5719..5823)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacattg attattgact agatcatcgc gtgaggctcc ggtgcccgtc agtgggcaga
       61 gcgcacatcg cccacagtcc ccgagaagtt ggggggaggg gtcggcaatt gaaccggtgc
      121 ctagagaagg tggcgcgggg taaactggga aagtgatgtc gtgtactggc tccgcctttt
      181 tcccgagggt gggggagaac cgtatataag tgcagtagtc gccgtgaacg ttctttttcg
      241 caacgggttt gccgccagaa cacaggtaag tgccgtgtgt ggttcccgcg ggcctggcct
      301 ctttacgggt tatggccctt gcgtgccttg aattacttcc acgcccctgg ctgcagtacg
      361 tgattcttga tcccgagctt cgggttggaa gtgggtggga gagttcgagg ccttgcgctt
      421 aaggagcccc ttcgcctcgt gcttgagttg aggcctggcc tgggcgctgg ggccgccgcg
      481 tgcgaatctg gtggcacctt cgcgcctgtc tcgctgcttt cgataagtct ctagccattt
      541 aaaatttttg atgacctgct gcgacgcttt ttttctggca agatagtctt gtaaatgcgg
      601 gccaagatct gcacactggt atttcggttt ttggggccgc gggcggcgac ggggcccgtg
      661 cgtcccagcg cacatgttcg gcgaggcggg gcctgcgagc gcggccaccg agaatcggac
      721 gggggtagtc tcaagctggc cggcctgctc tggtgcctgg cctcgcgccg ccgtgtatcg
      781 ccccgccctg ggcggcaagg ctggcccggt cggcaccagt tgcgtgagcg gaaagatggc
      841 cgcttcccgg ccctgctgca gggagctcaa aatggaggac gcggcgctcg ggagagcggg
      901 cgggtgagtc acccacacaa aggaaaaggg cctttccgtc ctcagccgtc gcttcatgtg
      961 actccacgga gtaccgggcg ccgtccaggc acctcgatta gttctcgagc ttttggagta
     1021 cgtcgtcttt aggttggggg gaggggtttt atgcgatgga gtttccccac actgagtggg
     1081 tggagactga agttaggcca gcttggcact tgatgtaatt ctccttggaa tttgcccttt
     1141 ttgagtttgg atcttggttc attctcaagc ctcagacagt ggttcaaagt ttttttcttc
     1201 catttcaggt gtcgtgagga attctgcagt cgacggtacc gcctacgcta gcgctaccgg
     1261 tcgccaccat ggtgagcaag ggcgaggagg ataacatggc catcatcaag gagttcatgc
     1321 gcttcaaggt gcacatggag ggctccgtga acggccacga gttcgagatc gagggcgagg
     1381 gcgagggccg cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggtggcc
     1441 ccctgccctt cgcctgggac atcctgtccc ctcagttcat gtacggctcc aaggcctacg
     1501 tgaagcaccc cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt
     1561 gggagcgcgt gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc
     1621 tgcaggacgg cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg
     1681 gccccgtaat gcagaagaag accatgggct gggaggcctc ctccgagcgg atgtaccccg
     1741 aggacggcgc cctgaagggc gagatcaagc agaggctgaa gctgaaggac ggcggccact
     1801 acgacgctga ggtcaagacc acctacaagg ccaagaagcc cgtgcagctg cccggcgcct
     1861 acaacgtcaa catcaagttg gacatcacct cccacaacga ggactacacc atcgtggaac
     1921 agtacgaacg cgccgagggc cgccactcca ccggcggcat ggacgagctg tacaagcccc
     1981 gggagggcag aggaagtctt ctaacatgcg gtgacgtgga ggagaatccc ggccctacta
     2041 gttgatgagg tggttcagga ggggcatgcg tgagcaaggg cgaggagctg ttcaccgggg
     2101 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     2161 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     2221 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     2281 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     2341 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     2401 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     2461 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     2521 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     2581 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     2641 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     2701 ccaacgagaa gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc
     2761 tcggcatgga cgagctgtac aagtaaagcg gccgcactcc tcaggtgcag gctgcctatc
     2821 agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga gatctttttc
     2881 cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact tctggctaat
     2941 aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa
     3001 ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg
     3061 caacatatgc catatgctgg ctgccatgaa caaaggtggc tataaagagg tcatcagtat
     3121 atgaaacagc cccctgctgt ccattcctta ttccatagaa aagccttgac ttgaggttag
     3181 atttttttta tattttgttt tgtgttattt ttttctttaa catccctaaa attttcctta
     3241 catgttttac tagccagatt tttcctcctc tcctgactac tcccagtcat agctgtccct
     3301 cttctcttat gaagatccct cgacctgcag cccaagcttg gcgtaatcat ggtcatagct
     3361 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     3421 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     3481 actgcccgct ttccagtcgg gaaacctgtc gtgccagcgg atccgcatct caattagtca
     3541 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3601 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctcg
     3661 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3721 aagctaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     3781 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     3841 tatcttatca tgtctggatc cgctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     3901 tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     3961 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     4021 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     4081 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     4141 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     4201 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     4261 ctttctccct tcgggaagcg tggcgctttc tcaatgctca cgctgtaggt atctcagttc
     4321 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     4381 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     4441 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     4501 gttcttgaag tggtggccta actacggcta cactagaagg acagtatttg gtatctgcgc
     4561 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     4621 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     4681 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     4741 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     4801 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     4861 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     4921 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     4981 tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag caataaacca
     5041 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     5101 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     5161 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     5221 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     5281 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     5341 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     5401 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     5461 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     5521 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     5581 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     5641 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     5701 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     5761 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     5821 gcgcacattt ccccgaaaag tgccacctg
//
