LOCUS       sequence-338860-        5852 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 5852)
  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 5852)
  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..5852
                     /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(3354..3370)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3354..3370)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3367..3389)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3378..3394
                     /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(3402..3408,3409..3426,3427..3432))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3447..3468
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        3527..3722
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      3573..3708
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    3728..3862
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3765..3784)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3819..3838
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3930..3947)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4101..4689)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4181..4200)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4860..5651,5652..5720))
                     /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     5483..5502
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5721..5825)
                     /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 cgaggagctt 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 ccatatgctg gctgccatga acaaaggttg gctataaaga ggtcatcagt
     3121 atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg acttgaggtt
     3181 agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta aaattttcct
     3241 tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc atagctgtcc
     3301 ctcttctctt atggagatcc ctcgacctgc agcccaagct tggcgtaatc atggtcatag
     3361 ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc
     3421 ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc
     3481 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc ggatccgcat ctcaattagt
     3541 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     3601 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     3661 cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     3721 aaaagctaac ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa
     3781 tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa
     3841 tgtatcttat catgtctgga tccgctgcat taatgaatcg gccaacgcgc ggggagaggc
     3901 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     3961 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     4021 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     4081 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     4141 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     4201 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     4261 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     4321 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     4381 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     4441 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     4501 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     4561 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     4621 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     4681 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     4741 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     4801 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     4861 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     4921 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     4981 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     5041 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     5101 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     5161 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     5221 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     5281 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     5341 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     5401 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     5461 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     5521 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     5581 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     5641 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     5701 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     5761 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     5821 ccgcgcacat ttccccgaaa agtgccacct gg
//
