LOCUS       sequence-468449-        5768 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the ZNF250-derived portion of 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 5768)
  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 5768)
  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: EW216 PGK-ZNF250(1-4)-deImmunLink-NZF(FLP-IN)
FEATURES             Location/Qualifiers
     source          1..5768
                     /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             1375..1404
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1460..1477)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1466..1690
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1862..1881)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2013..2060
                     /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             2068..3090
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2068..3090
                     /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    3220..3341
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3257..3276)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3311..3330
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3390..3406)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3390..3406)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3403..3425)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3414..3430
                     /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(3438..3444,3445..3462,3463..3468))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3483..3504
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3621..3638)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3792..4380)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3872..3891)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4551..5342,5343..5411))
                     /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     5174..5193
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5412..5516)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5591..5610)
                     /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 tatatgtgcg tagagtgcgg taaatgtttt gggcggagtt cacatctttt gcagcaccaa
      601 cgtattcaca caggcgagaa accctatgtt tgcagcgtgt gcggtaaggc gtttagccag
      661 tcctcagtcc tttccaaaca ccgccgcata catactggtg agaagccata tgaatgtaat
      721 gaatgtggca aggcttttag agtgagttca gatctcgctc aacatcataa gatccataca
      781 ggagagaagc cacacgaatg tttggagtgt cgaaaggctt ttactcagct cagtcacctc
      841 attcagcatc agagaattca cgctgctgca tgggagctag gaatgtctgg cggatctgag
      901 ggccagagcg acgagagagc cctgctggac cagctgcaca ccctgctgag caacaccgac
      961 gccaccggcc tggaggagat cgacagagcc ctgggcatcc ccgagctggt gaaccagggc
     1021 caggccctgg agcccaagca ggacggaggc tggggatcta tggccgagga attcgtgacc
     1081 ctgaaggacg tgggcatgga cttcaccctg ggcgactggg agcagctggg cctggagcag
     1141 ggcgacacct tctgggacac cgccctggac aactgccagg acctgttcct gctggacccc
     1201 cccggcggag gagacggcct tggtttggga cacgagaagt tccccagcga cctggacctg
     1261 gacatgttca acggcagcct ggagtgcgac atggagagca tcatcagaag cgagctgatg
     1321 gacgccgacg gcctggactt caacttcgac agctaggccg ctcgaggagg gcccgaacaa
     1381 aaactcatct cagaagagga tctgaatagc gccgtcgacc atcatcataa tcatcattga
     1441 gtttaaaccc gctgatcagc ctcgactgtg ccttctagtt gccagccatc tgttgtttgc
     1501 ccctcccccg tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa
     1561 aatgaggaaa ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg
     1621 gggcaggaca gcaaggggga ggattgggaa gacaatagca ggcatgctgg ggatgcggtg
     1681 ggctctatgg agatctatcg atgttctggg gatgcggtgg gctctatggc ttctgaggcg
     1741 gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc
     1801 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc
     1861 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct
     1921 ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa
     1981 aaacttgatt agggtgatgg ttcacgtacc tagaagttcc tattccgaag ttcctattct
     2041 ctagaaagta taggaacttc cttggccaaa aagcctgaac tcaccgcgac gtctgtcgag
     2101 aagtttctga tcgaaaagtt cgacagcgtc tccgacctga tgcagctctc ggagggcgaa
     2161 gaatctcgtg ctttcagctt cgatgtagga gggcgtggat atgtcctgcg ggtaaatagc
     2221 tgcgccgatg gtttctacaa agatcgttat gtttatcggc actttgcatc ggccgcgctc
     2281 ccgattccgg aagtgcttga cattggggaa ttcagcgaga gcctgaccta ttgcatctcc
     2341 cgccgtgcac agggtgtcac gttgcaagac ctgcctgaaa ccgaactgcc cgctgttctg
     2401 cagccggtcg cggaggccat ggatgcgatc gctgcggccg atcttagcca gacgagcggg
     2461 ttcggcccat tcggaccgca aggaatcggt caatacacta catggcgtga tttcatatgc
     2521 gcgattgctg atccccatgt gtatcactgg caaactgtga tggacgacac cgtcagtgcg
     2581 tccgtcgcgc aggctctcga tgagctgatg ctttgggccg aggactgccc cgaagtccgg
     2641 cacctcgtgc acgcggattt cggctccaac aatgtcctga cggacaatgg ccgcataaca
     2701 gcggtcattg actggagcga ggcgatgttc ggggattccc aatacgaggt cgccaacatc
     2761 ttcttctgga ggccgtggtt ggcttgtatg gagcagcaga cgcgctactt cgagcggagg
     2821 catccggagc ttgcaggatc gccgcggctc cgggcgtata tgctccgcat tggtcttgac
     2881 caactctatc agagcttggt tgacggcaat ttcgatgatg cagcttgggc gcagggtcga
     2941 tgcgacgcaa tcgtccgatc cggagccggg actgtcgggc gtacacaaat cgcccgcaga
     3001 agcgcggccg tctggaccga tggctgtgta gaagtactcg ccgatagtgg aaaccgacgc
     3061 cccagcactc gtccgagggc aaaggaatag cacgtactac gagatttcga ttccaccgcc
     3121 gccttctatg aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc
     3181 cagcgcgggg atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat
     3241 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     3301 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg
     3361 acctctagct agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat
     3421 ccgctcacaa ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc
     3481 taatgagtga gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     3541 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     3601 attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg
     3661 cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac
     3721 gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg
     3781 ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca
     3841 agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc
     3901 tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc
     3961 ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag
     4021 gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc
     4081 ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca
     4141 gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg
     4201 aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg
     4261 aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct
     4321 ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa
     4381 gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa
     4441 gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa
     4501 tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     4561 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     4621 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     4681 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     4741 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     4801 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     4861 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     4921 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     4981 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     5041 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     5101 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     5161 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     5221 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     5281 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     5341 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     5401 tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc
     5461 atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca
     5521 tttccccgaa aagtgccacc tgacgtcgac ggatcgggag atctcccgat cccctatggt
     5581 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtatctg ctccctgctt
     5641 gtgtgttgga ggtcgctgag tagtgcgcga gcaaaattta agctacaaca aggcaaggct
     5701 tgaccgacaa ttgcatgaag aatctgctta gggttaggcg ttttgcgctg cttcgtacgg
     5761 gccagata
//
