LOCUS       sequence-468426-        5927 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the ZNF35/ZNFIKZF1 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 5927)
  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 5927)
  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: EW415 PGK-ZNF35(5-8)-IKZF1(2-3)-realDeimmunLin...
FEATURES             Location/Qualifiers
     source          1..5927
                     /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"
     promoter        1402..1901
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1437..1460)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     1834..1853
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2922..2951
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(3007..3024)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3013..3237
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3409..3428)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3560..3607
                     /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             3615..4637
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3615..4637
                     /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    4767..4888
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4804..4823)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4858..4877
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4937..4953)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4937..4953)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4950..4972)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4961..4977
                     /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(4985..4991,4992..5009,5010..5015))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5030..5051
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5168..5185)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5339..5927)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5419..5438)
                     /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 cgcgttctac cgggtagggg aggcgctttt cccaaggcag tctggagcat
     1441 gcgctttagc agccccgctg ggcacttggc gctacacaag tggcctctgg cctcgcacac
     1501 attccacatc caccggtagg cgccaaccgg ctccgttctt tggtggcccc ttcgcgccac
     1561 cttctactcc tcccctagtc aggaagttcc cccccgcccc gcagctcgcg tcgtgcagga
     1621 cgtgacaaat ggaagtagca cgtctcatta gtctcgtgca gatggacagc accgctgagc
     1681 aatggaagcg ggtaggcctt tggggcagcg gccaatagca gctttgctcc ttcgctttct
     1741 gggctcagag gctgggaagg ggtgggtccg ggggcgggct caggggcggg ctcaggggcg
     1801 gggcgggcgc ccgaaggtcc tccggaggcc cggcattctg cacgcttcaa aagcgcacgt
     1861 ctgccgcgct gttctcctct tcctcatctc cgggcctttc gacctgcagc ccaagcttcg
     1921 ccaccatgta cgagtgtaat gagtgcggta agaccttcac acgatcttct aaccttattg
     1981 tgcatcaacg aatccataca ggcgagaagc cctttgcctg caatgactgt gggaaggctt
     2041 ttactcagag tgccaacttg atcgtgcatc agagaagcca tacgggtgag aagccctatg
     2101 aatgcaagga atgcggaaag gccttctcct gcttcagcca cctcattgta caccagcgca
     2161 tacacacggc tgagaagcca tacgattgct ctgaatgtgg aaaggccttt agtcagctgt
     2221 cttgcttgat tgtgcatcag agaattcaca cgggcgaacg ccccttccaa tgcaatcagt
     2281 gcggggcatc tttcactcag aaggggaact tactgcgcca tattaaattg cactctggtg
     2341 agaaaccgtt taaatgtcac ttatgcaact acgcctgcag gcgcagagat gccttgaccg
     2401 gccacctcag aacccatggc tggagcggag gcgatggcat gagcgagggc cagagcgacg
     2461 agagagccct gctggaccag ctgcacaccc tgctgagcaa caccgacgcc accggcctgg
     2521 aggagatcga cagagccctg ggcatccccg agctggtgaa ccagggccag gccctggagc
     2581 ccaagcagga cggaggctgg ggatctatgg ccgaggaatt cgtgaccctg aaggacgtgg
     2641 gcatggactt caccctgggc gactgggagc agctgggcct ggagcagggc gacaccttct
     2701 gggacaccgc cctggacaac tgccaggacc tgttcctgct ggacccccct ggcggaggag
     2761 acggccttgg tttgggacac gagaagttcc ccagcgacct ggacctggac atgttcaacg
     2821 gcagcctgga gtgcgacatg gagagcatca tcagaagcga gctgatggac gccgacggcc
     2881 tggacttcaa cttcgacagc taggccgctc gaggagggcc cgaacaaaaa ctcatctcag
     2941 aagaggatct gaatagcgcc gtcgaccatc atcataatca tcattgagtt taaacccgct
     3001 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     3061 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     3121 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     3181 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggaga
     3241 tctatcgatg ttctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     3301 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3361 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3421 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3481 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3541 gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag
     3601 gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     3661 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     3721 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     3781 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     3841 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     3901 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     3961 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     4021 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     4081 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     4141 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     4201 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     4261 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     4321 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     4381 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     4441 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     4501 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     4561 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     4621 cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa
     4681 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4741 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     4801 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4861 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     4921 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4981 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     5041 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5101 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5161 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5221 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5281 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5341 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5401 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5461 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5521 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5581 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5641 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5701 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5761 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5821 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5881 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaa
//
