LOCUS       sequence-468460-        6632 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding fluorescent reporter for PUF-9R binding which 
            expresses mCherry constitutively and EGFP upon stop codon 
            deamination due to ADAR-PUF9R binding .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6632)
  AUTHORS   Wolfsberg E, Paul J-P, Tycko J, Chen B, Bassik MC, Bintu L, Alizadeh
            AA, Gao XJ
  TITLE     Machine-Guided Dual-Objective Protein Engineering for Deimmunization
            and Therapeutic Functions
  JOURNAL   bioRxiv 2025.02.24.639918
REFERENCE   2  (bases 1 to 6632)
  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: "bioRxiv 
            2025.02.24.639918"
COMMENT     Sequence Label: EW265 SFFV-mCherry-SGc(stem-1(3xPUF9R-tar))cSG...
FEATURES             Location/Qualifiers
     source          1..6632
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(145..936,937..1005))
                     /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     768..787
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1006..1110)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1185..1204)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        1392..1799
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      1807..1816
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1813..2520
                     /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(1958..1976)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     2226..2245
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     CDS             2566..2631
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             2800..2865
                     /codon_start=1
                     /product="2A peptide from foot-and-mouth disease virus 
                     polyprotein"
                     /label=F2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="VKQTLNFDLLKLAGDVESNPGP"
     CDS             join(2872..2874,2875..3585)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             3601..3630
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3686..3703)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3692..3916
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4088..4107)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4239..4286
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             4294..5316
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4294..5316
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    5446..5567
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5483..5502)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5537..5556
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5616..5632)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5616..5632)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5629..5651)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5640..5656
                     /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(5664..5670,5671..5688,5689..5694))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5709..5730
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5847..5864)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6018..6606)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6098..6117)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa
       61 aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat
      121 atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc
      181 gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat
      241 acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
      301 ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc
      361 tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag
      421 ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg
      481 ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg
      541 atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag
      601 taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt
      661 catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga
      721 atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc
      781 acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc
      841 aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc
      901 ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
      961 cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca
     1021 atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat
     1081 ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctgacgt
     1141 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat
     1201 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc
     1261 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgccg ataaaataaa
     1321 agattttatt tagtctccag aaaaaggggg gaatgaaaga ccccacctgt aggtttggca
     1381 agctagctgc agtaacgcca ttttgcaagg catggaaaaa taccaaacca agaatagaga
     1441 agttcagatc aagggcgggt acatgaaaat agctaacgtt gggccaaaca ggatatctgc
     1501 ggtgagcagt ttcggccccg gcccggggcc aagaacagat ggtcaccgca gtttcggccc
     1561 cggcccgagg ccaagaacag atggtcccca gatatggccc aaccctcagc agtttcttaa
     1621 gacccatcag atgtttccag gctcccccaa ggacctgaaa tgaccctgcg ccttatttga
     1681 attaaccaat cagcctgctt ctcgcttctg ttcgcgcgct tctgcttccc gagctctata
     1741 aaagagctca caacccctca ctcggcgcgc cagtcctccg acagactgag tcgcccggga
     1801 agcttcgcca ccatggtgag caagggcgag gaggataaca tggccatcat caaggagttc
     1861 atgcgcttca aggtgcacat ggagggctcc gtgaacggcc acgagttcga gatcgagggc
     1921 gagggcgagg gccgccccta cgagggcacc cagaccgcca agctgaaggt gaccaagggt
     1981 ggccccctgc ccttcgcctg ggacatcctg tcccctcagt tcatgtacgg ctccaaggcc
     2041 tacgtgaagc accccgccga cattcccgac tacttgaagc tgtccttccc cgagggcttc
     2101 aagtgggagc gcgtgatgaa cttcgaggac ggcggcgtgg tgaccgtgac ccaggactcc
     2161 tccctgcagg acggcgagtt catctacaag gtgaagctgc gcggcaccaa cttcccctcc
     2221 gacggccccg taatgcagaa gaagaccatg ggctgggagg cctcctccga gcggatgtac
     2281 cccgaggacg gcgccctgaa gggcgagatc aagcagaggc tgaagctgaa ggacggcggc
     2341 cactacgacg ctgaggtcaa gaccacctac aaggccaaga agcccgtgca gctgcccggc
     2401 gcctacaacg tcaacatcaa gttggacatc acctcccaca acgaggacta caccatcgtg
     2461 gaacagtacg aacgcgccga gggccgccac tccaccggcg gcatggacga gctgtacaag
     2521 agcgacgcgt tggaagccag acacaaacag aaaattgtgg ctccggtgaa acagactttg
     2581 aattttgacc ttctgaagct ggcgggagac gtcgagtcca accccggtcc cggtaccgga
     2641 tcctccgttt aggtgggtgg aatagtaacg tactgttgta tagtacgtct cagactgttg
     2701 tatagtctgt ctggtatgtt gtatacgtac catagtatcc cacctaccca gacgaccggt
     2761 actagtgagg cacggcataa gcaaaagatc gtagccccag taaagcaaac actcaacttc
     2821 gatctactca aactcgcagg tgatgtggaa tctaatccag gaccctcgag tgtctcaaag
     2881 ggcgaggaac tttttacggg cgttgttccg atcctcgtcg aattggacgg ggacgttaat
     2941 gggcataaat tctcagttag tggtgagggt gagggggacg cgacatatgg gaaacttacg
     3001 ctgaagttca tatgtaccac cggaaaattg ccagtgccgt ggcctactct cgtcaccacc
     3061 ctgacatatg gtgttcagtg ttttagccgg tatccagacc atatgaaaca gcacgatttt
     3121 tttaaatccg ctatgcctga aggttacgta caggagcgaa cgatattttt taaagacgac
     3181 ggaaactaca aaactagagc cgaggtaaaa ttcgagggtg acactctcgt gaaccggatc
     3241 gaactgaagg gcatagattt taaggaggat ggcaatatac tgggccacaa gctcgaatac
     3301 aactataatt ctcataatgt ctatatcatg gcggacaaac agaaaaacgg catcaaggta
     3361 aactttaaaa tccgacacaa tattgaggac ggctctgtcc agcttgcgga ccattaccaa
     3421 cagaacacgc cgatagggga tggtccagta cttctccctg ataatcacta tttgtctact
     3481 cagtcagctc ttagcaagga cccaaacgaa aaacgggatc acatggtcct tcttgagttc
     3541 gtcactgcgg ctgggataac actcgggatg gacgaactgt ataagtaggc cggagggccc
     3601 gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcataatcat
     3661 cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     3721 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     3781 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     3841 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     3901 gcggtgggct ctatggagat ctatcgatgt tctggggatg cggtgggctc tatggcttct
     3961 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     4021 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     4081 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     4141 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     4201 cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc
     4261 tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct
     4321 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     4381 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     4441 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     4501 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     4561 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     4621 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     4681 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     4741 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     4801 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     4861 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     4921 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     4981 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     5041 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     5101 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     5161 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     5221 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     5281 cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc
     5341 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     5401 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     5461 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     5521 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     5581 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     5641 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     5701 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     5761 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5821 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5881 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5941 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     6001 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     6061 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     6121 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     6181 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     6241 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     6301 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     6361 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     6421 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     6481 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     6541 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     6601 tctcaagaag atcctttgat cttttctacg gg
//
