LOCUS       sequence-468442-        6331 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the ZNF35/ZNF250 zinc finger array attached by a 
            deimmunized linker to the NZF transcriptional activation domain, 
            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 6331)
  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 6331)
  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: EW369
            CMV-TO-ZNF35(5-8)-ZNF250(1-4)-deImmunLin...
FEATURES             Location/Qualifiers
     source          1..6331
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        384..587
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     538..558
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    589..607
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    610..628
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     638..662
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1933..1962
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2018..2035)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2024..2248
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2420..2439)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2571..2618
                     /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             2626..3648
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2626..3648
                     /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    3778..3899
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3815..3834)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3869..3888
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3948..3964)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3948..3964)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3961..3983)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3972..3988
                     /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(3996..4002,4003..4020,4021..4026))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4041..4062
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4179..4196)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4350..4938)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4430..4449)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5109..5900,5901..5969))
                     /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     5732..5751
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5970..6074)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6149..6168)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      421 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      481 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      541 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg
      601 atagagatct ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga
      661 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      721 gcctccggac tctagcgttt aaacttaagc ttcgccacca tgtacgagtg taatgagtgc
      781 ggtaagacct tcacacgatc ttctaacctt attgtgcatc aacgaatcca tacaggcgag
      841 aagccctttg cctgcaatga ctgtgggaag gcttttactc agagtgccaa cttgatcgtg
      901 catcagagaa gccatacggg tgagaagccc tatgaatgca aggaatgcgg aaaggccttc
      961 tcctgcttca gccacctcat tgtacaccag cgcatacaca cggctgagaa gccatacgat
     1021 tgctctgaat gtggaaaggc ctttagtcag ctgtcttgct tgattgtgca tcagagaatt
     1081 cacacgggcg aacgccccta tatgtgcgta gagtgcggta aatgttttgg gcggagttca
     1141 catcttttgc agcaccaacg tattcacaca ggcgagaaac cctatgtttg cagcgtgtgc
     1201 ggtaaggcgt ttagccagtc ctcagtcctt tccaaacacc gccgcataca tactggtgag
     1261 aagccatatg aatgtaatga atgtggcaag gcttttagag tgagttcaga tctcgctcaa
     1321 catcataaga tccatacagg agagaagcca cacgaatgtt tggagtgtcg aaaggctttt
     1381 actcagctca gtcacctcat tcagcatcag agaattcacg ctgctgcatg ggagctagga
     1441 atgtctggcg gatctgaggg ccagagcgac gagagagccc tgctggacca gctgcacacc
     1501 ctgctgagca acaccgacgc caccggcctg gaggagatcg acagagccct gggcatcccc
     1561 gagctggtga accagggcca ggccctggag cccaagcagg acggaggctg gggatctatg
     1621 gccgaggaat tcgtgaccct gaaggacgtg ggcatggact tcaccctggg cgactgggag
     1681 cagctgggcc tggagcaggg cgacaccttc tgggacaccg ccctggacaa ctgccaggac
     1741 ctgttcctgc tggacccccc cggcggagga gacggccttg gtttgggaca cgagaagttc
     1801 cccagcgacc tggacctgga catgttcaac ggcagcctgg agtgcgacat ggagagcatc
     1861 atcagaagcg agctgatgga cgccgacggc ctggacttca acttcgacag ctaggccgct
     1921 cgaggagggc ccgaacaaaa actcatctca gaagaggatc tgaatagcgc cgtcgaccat
     1981 catcataatc atcattgagt ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc
     2041 cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg tgccactccc
     2101 actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct
     2161 attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga caatagcagg
     2221 catgctgggg atgcggtggg ctctatggag atctatcgat gttctgggga tgcggtgggc
     2281 tctatggctt ctgaggcgga aagaaccagc tggggctcta gggggtatcc ccacgcgccc
     2341 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt
     2401 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc
     2461 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta
     2521 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtaccta gaagttccta
     2581 ttccgaagtt cctattctct agaaagtata ggaacttcct tggccaaaaa gcctgaactc
     2641 accgcgacgt ctgtcgagaa gtttctgatc gaaaagttcg acagcgtctc cgacctgatg
     2701 cagctctcgg agggcgaaga atctcgtgct ttcagcttcg atgtaggagg gcgtggatat
     2761 gtcctgcggg taaatagctg cgccgatggt ttctacaaag atcgttatgt ttatcggcac
     2821 tttgcatcgg ccgcgctccc gattccggaa gtgcttgaca ttggggaatt cagcgagagc
     2881 ctgacctatt gcatctcccg ccgtgcacag ggtgtcacgt tgcaagacct gcctgaaacc
     2941 gaactgcccg ctgttctgca gccggtcgcg gaggccatgg atgcgatcgc tgcggccgat
     3001 cttagccaga cgagcgggtt cggcccattc ggaccgcaag gaatcggtca atacactaca
     3061 tggcgtgatt tcatatgcgc gattgctgat ccccatgtgt atcactggca aactgtgatg
     3121 gacgacaccg tcagtgcgtc cgtcgcgcag gctctcgatg agctgatgct ttgggccgag
     3181 gactgccccg aagtccggca cctcgtgcac gcggatttcg gctccaacaa tgtcctgacg
     3241 gacaatggcc gcataacagc ggtcattgac tggagcgagg cgatgttcgg ggattcccaa
     3301 tacgaggtcg ccaacatctt cttctggagg ccgtggttgg cttgtatgga gcagcagacg
     3361 cgctacttcg agcggaggca tccggagctt gcaggatcgc cgcggctccg ggcgtatatg
     3421 ctccgcattg gtcttgacca actctatcag agcttggttg acggcaattt cgatgatgca
     3481 gcttgggcgc agggtcgatg cgacgcaatc gtccgatccg gagccgggac tgtcgggcgt
     3541 acacaaatcg cccgcagaag cgcggccgtc tggaccgatg gctgtgtaga agtactcgcc
     3601 gatagtggaa accgacgccc cagcactcgt ccgagggcaa aggaatagca cgtactacga
     3661 gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt tttccgggac
     3721 gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc ccaccccaac
     3781 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     3841 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     3901 catgtctgta taccgtcgac ctctagctag agcttggcgt aatcatggtc atagctgttt
     3961 cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag
     4021 tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg
     4081 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     4141 gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc
     4201 tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc
     4261 acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg
     4321 aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat
     4381 cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag
     4441 gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
     4501 tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg
     4561 tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt
     4621 cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac
     4681 gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc
     4741 ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag aacagtattt
     4801 ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
     4861 ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc
     4921 agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg
     4981 aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag
     5041 atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg
     5101 tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt
     5161 tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca
     5221 tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca
     5281 gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc
     5341 tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt
     5401 ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg
     5461 gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc
     5521 aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg
     5581 ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga
     5641 tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga
     5701 ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta
     5761 aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg
     5821 ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact
     5881 ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata
     5941 agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt
     6001 tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa
     6061 ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtcgacgg atcgggagat
     6121 ctcccgatcc cctatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     6181 agtatctgct ccctgcttgt gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag
     6241 ctacaacaag gcaaggcttg accgacaatt gcatgaagaa tctgcttagg gttaggcgtt
     6301 ttgcgctgct tcgtacgggc cagatatacg c
//
