LOCUS       sequence-468459-        6104 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 PGK promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6104)
  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 6104)
  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: EW196
            PGK-ZNF35(5-8)-ZNF250(1-4)-deImmunLink-N...
FEATURES             Location/Qualifiers
     source          1..6104
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        14..513
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(49..72)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     446..465
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             1711..1740
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1796..1813)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1802..2026
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2198..2217)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2349..2396
                     /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             2404..3426
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2404..3426
                     /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    3556..3677
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3593..3612)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3647..3666
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3726..3742)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3726..3742)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3739..3761)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3750..3766
                     /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(3774..3780,3781..3798,3799..3804))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3819..3840
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3957..3974)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4128..4716)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4208..4227)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4887..5678,5679..5747))
                     /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     5510..5529
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5748..5852)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5927..5946)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tacgcgttct accgggtagg ggaggcgctt ttcccaaggc agtctggagc atgcgcttta
       61 gcagccccgc tgggcacttg gcgctacaca agtggcctct ggcctcgcac acattccaca
      121 tccaccggta ggcgccaacc ggctccgttc tttggtggcc ccttcgcgcc accttctact
      181 cctcccctag tcaggaagtt cccccccgcc ccgcagctcg cgtcgtgcag gacgtgacaa
      241 atggaagtag cacgtctcat tagtctcgtg cagatggaca gcaccgctga gcaatggaag
      301 cgggtaggcc tttggggcag cggccaatag cagctttgct ccttcgcttt ctgggctcag
      361 aggctgggaa ggggtgggtc cgggggcggg ctcaggggcg ggctcagggg cggggcgggc
      421 gcccgaaggt cctccggagg cccggcattc tgcacgcttc aaaagcgcac gtctgccgcg
      481 ctgttctcct cttcctcatc tccgggcctt tcgacctgca gcccaagctt cgccaccatg
      541 tacgagtgta atgagtgcgg taagaccttc acacgatctt ctaaccttat tgtgcatcaa
      601 cgaatccata caggcgagaa gccctttgcc tgcaatgact gtgggaaggc ttttactcag
      661 agtgccaact tgatcgtgca tcagagaagc catacgggtg agaagcccta tgaatgcaag
      721 gaatgcggaa aggccttctc ctgcttcagc cacctcattg tacaccagcg catacacacg
      781 gctgagaagc catacgattg ctctgaatgt ggaaaggcct ttagtcagct gtcttgcttg
      841 attgtgcatc agagaattca cacgggcgaa cgcccctata tgtgcgtaga gtgcggtaaa
      901 tgttttgggc ggagttcaca tcttttgcag caccaacgta ttcacacagg cgagaaaccc
      961 tatgtttgca gcgtgtgcgg taaggcgttt agccagtcct cagtcctttc caaacaccgc
     1021 cgcatacata ctggtgagaa gccatatgaa tgtaatgaat gtggcaaggc ttttagagtg
     1081 agttcagatc tcgctcaaca tcataagatc catacaggag agaagccaca cgaatgtttg
     1141 gagtgtcgaa aggcttttac tcagctcagt cacctcattc agcatcagag aattcacgct
     1201 gctgcatggg agctaggaat gtctggcgga tctgagggcc agagcgacga gagagccctg
     1261 ctggaccagc tgcacaccct gctgagcaac accgacgcca ccggcctgga ggagatcgac
     1321 agagccctgg gcatccccga gctggtgaac cagggccagg ccctggagcc caagcaggac
     1381 ggaggctggg gatctatggc cgaggaattc gtgaccctga aggacgtggg catggacttc
     1441 accctgggcg actgggagca gctgggcctg gagcagggcg acaccttctg ggacaccgcc
     1501 ctggacaact gccaggacct gttcctgctg gacccccccg gcggaggaga cggccttggt
     1561 ttgggacacg agaagttccc cagcgacctg gacctggaca tgttcaacgg cagcctggag
     1621 tgcgacatgg agagcatcat cagaagcgag ctgatggacg ccgacggcct ggacttcaac
     1681 ttcgacagct aggccgctcg aggagggccc gaacaaaaac tcatctcaga agaggatctg
     1741 aatagcgccg tcgaccatca tcataatcat cattgagttt aaacccgctg atcagcctcg
     1801 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     1861 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     1921 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     1981 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggagat ctatcgatgt
     2041 tctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     2101 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2161 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     2221 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2281 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2341 cgtacctaga agttcctatt ccgaagttcc tattctctag aaagtatagg aacttccttg
     2401 gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac
     2461 agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
     2521 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat
     2581 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt
     2641 ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg
     2701 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat
     2761 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
     2821 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat
     2881 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag
     2941 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc
     3001 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg
     3061 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
     3121 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg
     3181 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac
     3241 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga
     3301 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc
     3361 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag
     3421 gaatagcacg tactacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     3481 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     3541 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3601 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3661 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     3721 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     3781 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     3841 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     3901 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     3961 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4021 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4081 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4141 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4201 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4261 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4321 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4381 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4441 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4501 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4561 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4621 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4681 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4741 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     4801 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     4861 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     4921 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     4981 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5041 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5101 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5161 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5221 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5281 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5341 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5401 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5461 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5521 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5581 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5641 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5701 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     5761 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     5821 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     5881 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     5941 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     6001 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     6061 tgcttagggt taggcgtttt gcgctgcttc gtacgggcca gata
//
