LOCUS       sequence-468415-        6391 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding free ADAR2dd 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 6391)
  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 6391)
  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: EW446 CMV-TO-ADAR2dd(E488Q, T501A) (FLP-IN)
FEATURES             Location/Qualifiers
     source          1..6391
                     /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             3386..3415
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3471..3488)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3477..3701
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3873..3892)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4024..4071
                     /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             4079..5101
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4079..5101
                     /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    5231..5352
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5268..5287)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5322..5341
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5401..5417)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5401..5417)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5414..5436)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5425..5441
                     /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(5449..5455,5456..5473,5474..5479))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5494..5515
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5632..5649)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5803..6391)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5883..5902)
                     /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 gccctgagcc gctcgaggag ggcccgaaca aaaactcatc tcagaagagg atctgaatag
     3421 cgccgtcgac catcatcata atcatcattg agtttaaacc cgctgatcag cctcgactgt
     3481 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     3541 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     3601 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     3661 agacaatagc aggcatgctg gggatgcggt gggctctatg gagatctatc gatgttctgg
     3721 ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta
     3781 tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt
     3841 gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct
     3901 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg
     3961 atttagtgct ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac
     4021 ctagaagttc ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa
     4081 aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt
     4141 ctccgacctg atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg
     4201 agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta
     4261 tgtttatcgg cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga
     4321 attcagcgag agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga
     4381 cctgcctgaa accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat
     4441 cgctgcggcc gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg
     4501 tcaatacact acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg
     4561 gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat
     4621 gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa
     4681 caatgtcctg acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt
     4741 cggggattcc caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat
     4801 ggagcagcag acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct
     4861 ccgggcgtat atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa
     4921 tttcgatgat gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg
     4981 gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt
     5041 agaagtactc gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata
     5101 gcacgtacta cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat
     5161 cgttttccgg gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt
     5221 cgcccacccc aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac
     5281 aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat
     5341 caatgtatct tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg
     5401 gtcatagctg tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc
     5461 cggaagcata aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc
     5521 gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat
     5581 cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac
     5641 tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt
     5701 aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca
     5761 gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc
     5821 ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact
     5881 ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct
     5941 gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag
     6001 ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca
     6061 cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa
     6121 cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc
     6181 gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag
     6241 aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg
     6301 tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca
     6361 gcagattacg cgcagaaaaa aaggatctca a
//
