LOCUS       sequence-468434-        6232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the ZNF35/ZNF250 zinc finger array attached by a 
            deimmunized linker to FKBP1A, 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 6232)
  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 6232)
  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: EW489 CMV-TO-ZNF35(5-8)-ZNF250(1-4)-(GGS)x8[G2...
FEATURES             Location/Qualifiers
     source          1..6232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        291..494
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     445..465
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    496..514
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    517..535
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     545..569
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1399..1722
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             1741..1770
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1826..1843)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1832..2056
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2228..2247)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2379..2426
                     /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             2434..3456
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2434..3456
                     /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    3586..3707
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3623..3642)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3677..3696
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3756..3772)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3756..3772)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3769..3791)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3780..3796
                     /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(3804..3810,3811..3828,3829..3834))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3849..3870
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3987..4004)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4158..4746)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4238..4257)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4917..5708,5709..5777))
                     /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     5540..5559
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5778..5882)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5957..5976)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6143..6232,1..290)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
       61 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      121 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      181 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      241 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      301 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      361 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      421 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      481 atataagcag agctctccct atcagtgata gagatctccc tatcagtgat agagatcgtc
      541 gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
      601 tccatagaag acaccgggac cgatccagcc tccggactct agcgtttaaa cttaagcttc
      661 gccaccatgt acgagtgtaa tgagtgcggt aagaccttca cacgatcttc taaccttatt
      721 gtgcatcaac gaatccatac aggcgagaag ccctttgcct gcaatgactg tgggaaggct
      781 tttactcaga gtgccaactt gatcgtgcat cagagaagcc atacgggtga gaagccctat
      841 gaatgcaagg aatgcggaaa ggccttctcc tgcttcagcc acctcattgt acaccagcgc
      901 atacacacgg ctgagaagcc atacgattgc tctgaatgtg gaaaggcctt tagtcagctg
      961 tcttgcttga ttgtgcatca gagaattcac acgggcgaac gcccctatat gtgcgtagag
     1021 tgcggtaaat gttttgggcg gagttcacat cttttgcagc accaacgtat tcacacaggc
     1081 gagaaaccct atgtttgcag cgtgtgcggt aaggcgttta gccagtcctc agtcctttcc
     1141 aaacaccgcc gcatacatac tggtgagaag ccatatgaat gtaatgaatg tggcaaggct
     1201 tttagagtga gttcagatct cgctcaacat cataagatcc atacaggaga gaagccacac
     1261 gaatgtttgg agtgtcgaaa ggcttttact cagctcagtc acctcattca gcatcagaga
     1321 attcacggcg taagctgggg actactgggc tcttgggaca gccttggatc tggcatgagc
     1381 ataggctcta tgcaacaggg agtgcaggtg gaaaccatct ccccaggaga cgggcgcacc
     1441 ttccccaagc gcggccagac ctgcgtggtg cactacaccg ggatgcttga agatggaaag
     1501 aaatttgatt cctcccggga cagaaacaag ccctttaagt ttatgctagg caagcaggag
     1561 gtgatccgag gctgggaaga aggggttgcc cagatgagtg tgggtcagag agccaaactg
     1621 actatatctc cagattatgc ctatggtgcc actgggcacc caggcatcat cccaccacat
     1681 gccactctcg tcttcgatgt ggagcttcta aaactggaat aggccgctcg aggagggccc
     1741 gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcataatcat
     1801 cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     1861 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     1921 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     1981 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     2041 gcggtgggct ctatggagat ctatcgatgt tctggggatg cggtgggctc tatggcttct
     2101 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2161 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2221 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2281 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2341 cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc
     2401 tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct
     2461 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     2521 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     2581 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     2641 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     2701 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     2761 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     2821 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     2881 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     2941 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     3001 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     3061 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     3121 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     3181 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     3241 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     3301 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     3361 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     3421 cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc
     3481 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     3541 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     3601 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3661 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     3721 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3781 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3841 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3901 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3961 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     4021 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     4081 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4141 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4201 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4261 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4321 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4381 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4441 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4501 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4561 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4621 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4681 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4741 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4801 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4861 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4921 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4981 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     5041 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     5101 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5161 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5221 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5281 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5341 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5401 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5461 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5521 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5581 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5641 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5701 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5761 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5821 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5881 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
     5941 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
     6001 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
     6061 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
     6121 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
     6181 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact ta
//
