LOCUS       sequence-468401-        5992 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses NZF transcriptional activation domain attached by a GS 
            linker to the mTOR FRB rapamycin binding domain with the T098L 
            mutation, 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 5992)
  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 5992)
  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: EW392 CMV-TO-NZF-(GGS)x8-FRB(T2098L) (FLP-IN)
FEATURES             Location/Qualifiers
     source          1..5992
                     /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"
     regulatory      661..670
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1501..1530
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1586..1603)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1592..1816
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1988..2007)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2139..2186
                     /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             2194..3216
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2194..3216
                     /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    3346..3467
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3383..3402)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3437..3456
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3516..3532)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3516..3532)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3529..3551)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3540..3556
                     /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(3564..3570,3571..3588,3589..3594))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3609..3630
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3747..3764)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3918..4506)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3998..4017)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4677..5468,5469..5537))
                     /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     5300..5319
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5538..5642)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5717..5736)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(5903..5992,1..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 gccaccatgg agggccagag cgacgagaga gccctgctgg accagctgca caccctgctg
      721 agcaacaccg acgccaccgg cctggaggag atcgacagag ccctgggcat ccccgagctg
      781 gtgaaccagg gccaggccct ggagcccaag caggacggag gctggggatc tatggccgag
      841 gaattcgtga ccctgaagga cgtgggcatg gacttcaccc tgggcgactg ggagcagctg
      901 ggcctggagc agggcgacac cttctgggac accgccctgg acaactgcca ggacctgttc
      961 ctgctggacc cccccggcgg aggagacggc cttggtttgg gacacgagaa gttccccagc
     1021 gacctggacc tggacatgtt caacggcagc ctggagtgcg acatggagag catcatcaga
     1081 agcgagctga tggacgccga cggcctggac ttcaacttcg acagcgctgc tgcaggcgga
     1141 agcggcggat ctggaggctc tggcggaagc ggcggatctg gcggaagcgg aggctctggc
     1201 ggatctgaga tgtggcatga aggcctggaa gaggcatctc gtttgtactt tggggaaagg
     1261 aacgtgaaag gcatgtttga ggtgctggag cccttgcatg ctatgatgga acggggaccc
     1321 cagactctga aggaaacatc ctttaatcag gcctatggtc gagatttaat ggaggcccaa
     1381 gagtggtgca ggaagtacat gaaatcaggg aatgtcaagg acctcctcca agcctgggac
     1441 ctctattatc atgtgttccg acgaatctca aagactagat aggccgctcg aggagggccc
     1501 gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcataatcat
     1561 cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     1621 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     1681 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     1741 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     1801 gcggtgggct ctatggagat ctatcgatgt tctggggatg cggtgggctc tatggcttct
     1861 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     1921 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     1981 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2041 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2101 cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc
     2161 tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct
     2221 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     2281 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     2341 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     2401 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     2461 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     2521 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     2581 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     2641 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     2701 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     2761 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     2821 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     2881 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     2941 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     3001 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     3061 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     3121 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     3181 cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc
     3241 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     3301 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     3361 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3421 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     3481 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3541 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3601 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3661 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3721 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3781 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3841 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     3901 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     3961 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4021 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4081 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4141 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4201 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4261 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4321 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4381 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4441 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4501 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4561 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4621 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4681 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4741 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4801 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4861 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     4921 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     4981 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5041 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5101 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5161 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5221 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5281 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5341 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5401 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5461 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5521 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5581 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5641 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
     5701 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
     5761 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
     5821 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
     5881 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
     5941 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact ta
//
