LOCUS       sequence-468402-        6241 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the ZNF35/ZNF250 zinc finger array attached by a GS
            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 6241)
  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 6241)
  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: EW394 CMV-TO-ZNF35(5-8)-ZNF250(1-4)-(GGS)x8-FK...
FEATURES             Location/Qualifiers
     source          1..6241
                     /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             1408..1731
                     /codon_start=1
                     /product="human FK506-binding protein FKBP12"
                     /label=FKBP
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKFDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             1750..1779
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1835..1852)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1841..2065
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2237..2256)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2388..2435
                     /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             2443..3465
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2443..3465
                     /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    3595..3716
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3632..3651)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3686..3705
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3765..3781)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3765..3781)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3778..3800)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3789..3805
                     /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(3813..3819,3820..3837,3838..3843))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3858..3879
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3996..4013)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4167..4755)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4247..4266)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4926..5717,5718..5786))
                     /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     5549..5568
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5787..5891)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5966..5985)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6152..6241,1..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 attcacgctg ctgcaggcgg aagcggcgga tctggaggct ctggcggaag cggcggatct
     1381 ggcggaagcg gaggctctgg cggatctgga gtgcaggtgg aaaccatctc cccaggagac
     1441 gggcgcacct tccccaagcg cggccagacc tgcgtggtgc actacaccgg gatgcttgaa
     1501 gatggaaaga aatttgattc ctcccgggac agaaacaagc cctttaagtt tatgctaggc
     1561 aagcaggagg tgatccgagg ctgggaagaa ggggttgccc agatgagtgt gggtcagaga
     1621 gccaaactga ctatatctcc agattatgcc tatggtgcca ctgggcaccc aggcatcatc
     1681 ccaccacatg ccactctcgt cttcgatgtg gagcttctaa aactggaata ggccgctcga
     1741 ggagggcccg aacaaaaact catctcagaa gaggatctga atagcgccgt cgaccatcat
     1801 cataatcatc attgagttta aacccgctga tcagcctcga ctgtgccttc tagttgccag
     1861 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     1921 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     1981 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagacaa tagcaggcat
     2041 gctggggatg cggtgggctc tatggagatc tatcgatgtt ctggggatgc ggtgggctct
     2101 atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt
     2161 agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc
     2221 agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc
     2281 tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg
     2341 cacctcgacc ccaaaaaact tgattagggt gatggttcac gtacctagaa gttcctattc
     2401 cgaagttcct attctctaga aagtatagga acttccttgg ccaaaaagcc tgaactcacc
     2461 gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga cctgatgcag
     2521 ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg tggatatgtc
     2581 ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta tcggcacttt
     2641 gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag cgagagcctg
     2701 acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa
     2761 ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc ggccgatctt
     2821 agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata cactacatgg
     2881 cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac tgtgatggac
     2941 gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac
     3001 tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt cctgacggac
     3061 aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga ttcccaatac
     3121 gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca gcagacgcgc
     3181 tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc gtatatgctc
     3241 cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga tgatgcagct
     3301 tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca
     3361 caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt actcgccgat
     3421 agtggaaacc gacgccccag cactcgtccg agggcaaagg aatagcacgt actacgagat
     3481 ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc
     3541 ggctggatga tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg
     3601 tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa
     3661 gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat
     3721 gtctgtatac cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct
     3781 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     3841 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     3901 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     3961 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4021 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4081 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4141 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4201 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4261 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4321 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4381 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     4441 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     4501 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     4561 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     4621 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     4681 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     4741 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     4801 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     4861 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     4921 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     4981 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5041 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5101 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5161 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5221 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     5281 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     5341 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     5401 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     5461 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     5521 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     5581 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     5641 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     5701 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     5761 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     5821 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     5881 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
     5941 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
     6001 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
     6061 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
     6121 cgctgcttcg tacgggccag atatacgcgt tgacattgat tattgactag ttattaatag
     6181 taatcaatta cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt
     6241 a
//
