LOCUS       sequence-468464-        7597 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding ADAR2dd attached to PUF-9R with deimmunizing and 
            function-restoring mutations under control of CMV promoter with two 
            TetR binding sites.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7597)
  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 7597)
  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: EW482 CMV-TO-ADAR2dd(E488Q,
            T501A)-PUF-9R-MARI...
FEATURES             Location/Qualifiers
     source          1..7597
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(171..962,963..1031))
                     /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     794..813
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1032..1136)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(1211..1230)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        1397..1776
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1777..1980
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1931..1951
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    1982..2000
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    2003..2021
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     2031..2055
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             4592..4621
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(4677..4694)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4683..4907
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(5079..5098)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    5230..5277
                     /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             5285..6307
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             5285..6307
                     /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    6437..6558
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6474..6493)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6528..6547
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6607..6623)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6607..6623)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6620..6642)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6631..6647
                     /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(6655..6661,6662..6679,6680..6685))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6700..6721
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6838..6855)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7009..7597)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7089..7108)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
       61 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
      121 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
      181 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
      241 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
      301 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
      361 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
      421 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
      481 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
      541 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
      601 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
      661 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
      721 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
      781 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
      841 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
      901 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
      961 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     1021 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     1081 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     1141 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     1201 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     1261 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     1321 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgtacgg
     1381 gccagatata cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg
     1441 tcattagttc atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg
     1501 cctggctgac cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata
     1561 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     1621 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     1681 ggtaaatggc ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg
     1741 cagtacatct acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc
     1801 aatgggcgtg gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc
     1861 aatgggagtt tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc
     1921 gccccattga cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct
     1981 ctccctatca gtgatagaga tctccctatc agtgatagag atcgtcgacg agctcgttta
     2041 gtgaaccgtc agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac
     2101 cgggaccgat ccagcctccg gactctagcg tttaaactta agcttcgcca ccatgttgca
     2161 cttggatcaa acgccatctc gccagcctat tcccagtgag ggtcttcagc tgcatttacc
     2221 gcaggtttta gctgacgctg tctcacgcct ggtcctgggt aagtttggtg acctgaccga
     2281 caacttctcc tcccctcacg ctcgcagaaa agtgctggct ggagtcgtca tgacaacagg
     2341 cacagatgtt aaagatgcca aggtgataag tgtttctaca ggaacaaaat gtattaatgg
     2401 tgaatacatg agtgatcgtg gccttgcatt aaatgactgc catgcagaaa taatatctcg
     2461 gagatccttg ctcagatttc tttatacaca acttgagctt tacttaaata acaaagatga
     2521 tcaaaaaaga tccatctttc agaaatcaga gcgagggggg tttaggctga aggagaatgt
     2581 ccagtttcat ctgtacatca gcacctctcc ctgtggagat gccagaatct tctcaccaca
     2641 tgagccaatc ctggaagaac cagcagatag acacccaaat cgtaaagcaa gaggacagct
     2701 acggaccaaa atagagtctg gtcaggggac gattccagtg cgctccaatg cgagcatcca
     2761 agcgtgggac ggggtgctgc aaggggagcg gctgctcacc atgtcctgca gtgacaagat
     2821 tgcacgctgg aacgtggtgg gcatccaggg atccctgctc agcattttcg tggagcccat
     2881 ttacttctcg agcatcatcc tgggcagcct ttaccacggg gaccaccttt ccagggccat
     2941 gtaccagcgg atctccaaca tagaggacct gccacctctc tacaccctca acaagccttt
     3001 gctcagtggc atcagcaatg cagaagcacg gcagccaggg aaggccccca acttcagtgt
     3061 caactggacg gtaggcgact ccgctattga ggtcatcaac gccacgactg ggaaggatga
     3121 gctgggccgc gcgtcccgcc tgtgtaagca cgcgttgtac tgtcgctgga tgcgtgtgca
     3181 cggcaaggtt ccctcccact tactacgctc caagattacc aaacccaacg tgtaccatga
     3241 gtccaagctg gcggcaaagg agtaccaggc cgccaaggcg cgtctgttca cagccttcat
     3301 caaggcgggg ctgggggcct gggtggagaa gcccaccgag caggaccagt tctcactcac
     3361 gcccggtacc ggtggcggaa ccggtggcgg ggctggcagg agtaggcttc tggaagactt
     3421 taggaataat cgctatccga atctccaact ccgtgaaatt gccggccata tcatggagtt
     3481 ttcacaagac caacatggga gccggttcat tcagctgaag ttagagagag ctaccccagc
     3541 agaacgacag ctcgtgttta acgaaatcct gcaagcggca tatcaactga tggtagacgt
     3601 tttcggtaac tacgtgattc aaaagttctt tgaatttgga tctcttgaac agaaactcgc
     3661 tcttgccgaa cgtatacggg gccacgtatt atccctcgcc ttacagatgt acggttgtcg
     3721 ggttattcag aaagctcttg aattcatccc gtcagaccag cagaatgaaa tggtccgaga
     3781 attgcatcaa catacggaac agcttgtaca agaccaatat ggtaactatg tcatccagca
     3841 cgtattggag cacggaagac cggaagataa atcaaagatt gttgccgaga taagagggaa
     3901 cgtacttgtc ctgagtcagc ataagttcgc aagtaatgtc gtcgagaagt gtgtgaccca
     3961 tgcgagcaga actgaacggg cagtgcttat cgacgaagtg tgcacgatga acgatggtcc
     4021 tcactcagcg ctgtatacaa tgatgaaaga ccaatacgcg aattatgtgg tgcagaagat
     4081 gatagatgtg gctgaaccag gccaaaggaa aatttgtatg caggaactcc atcaacacac
     4141 cgagcagctg gttcaagacc aatacgggaa ctatgtgatt caacacgtcc tcgaacacgg
     4201 acgacctgaa gataaaagta aaatcgtcgc ggaaatccgt ggtaatgtgc tcgttctttc
     4261 ccaacataag tttgcaagca acgtcgtgga aaagtgtgtg acacatgcct caaggacaga
     4321 aagggcggtc ctcattgatg aagtctgcac aatgaacgat ggcccccata gcgcattgta
     4381 tactatgatg aaagatcaat atgcgaacta cgttgtccag aaaatgattg acgtggctga
     4441 gccaggacaa aggaaaatcg tcatgcacaa aatcagacca cacatcgcaa ctctcaggaa
     4501 atacacatat ggtaaacaca tacttgcaaa gctggagaag tattacatga agaatggggt
     4561 agacctgggc tgagccgctc gaggagggcc cgaacaaaaa ctcatctcag aagaggatct
     4621 gaatagcgcc gtcgaccatc atcataatca tcattgagtt taaacccgct gatcagcctc
     4681 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     4741 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     4801 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     4861 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggaga tctatcgatg
     4921 ttctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     4981 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     5041 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     5101 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     5161 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     5221 acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag gaacttcctt
     5281 ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg aaaagttcga
     5341 cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt tcagcttcga
     5401 tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt tctacaaaga
     5461 tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag tgcttgacat
     5521 tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg gtgtcacgtt
     5581 gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg aggccatgga
     5641 tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg gaccgcaagg
     5701 aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc cccatgtgta
     5761 tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg ctctcgatga
     5821 gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg cggatttcgg
     5881 ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact ggagcgaggc
     5941 gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc cgtggttggc
     6001 ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg caggatcgcc
     6061 gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga gcttggttga
     6121 cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg tccgatccgg
     6181 agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct ggaccgatgg
     6241 ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc cgagggcaaa
     6301 ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa ggttgggctt
     6361 cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc tcatgctgga
     6421 gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     6481 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     6541 actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga gcttggcgta
     6601 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     6661 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     6721 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     6781 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     6841 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     6901 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     6961 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     7021 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     7081 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     7141 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     7201 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     7261 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     7321 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     7381 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     7441 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     7501 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     7561 caagcagcag attacgcgca gaaaaaaagg atctcaa
//
