LOCUS       sequence-468457-        6112 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding Gal4-NZF transcription factor with deimmunized 
            linkers between the component domains of NZF, 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 6112)
  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 6112)
  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: EW152 CMV-TO-Gal4-NZF[Mutated Linkers] (FLP-IN)
FEATURES             Location/Qualifiers
     source          1..6112
                     /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             667..1107
                     /codon_start=1
                     /gene="Saccharomyces cerevisiae GAL4 (truncated)"
                     /product="DNA binding domain of the GAL4 transcriptional 
                     activator"
                     /label=GAL4 DNA binding domain
                     /translation="MKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK
                     RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNK
                     DAVTDRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVS"
     CDS             1621..1650
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1706..1723)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1712..1936
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2108..2127)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2259..2306
                     /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             2314..3336
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2314..3336
                     /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    3466..3587
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3503..3522)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3557..3576
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3636..3652)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3636..3652)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3649..3671)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3660..3676
                     /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(3684..3690,3691..3708,3709..3714))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3729..3750
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3867..3884)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4038..4626)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4118..4137)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4797..5588,5589..5657))
                     /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     5420..5439
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5658..5762)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5837..5856)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6023..6112,1..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 gccaccatga agctgctgag cagcatcgag caggcctgtg acatctgccg gctgaagaaa
      721 ctgaagtgca gcaaagaaaa gcccaagtgc gccaagtgcc tgaagaacaa ctgggagtgc
      781 cggtacagcc ccaagaccaa gagaagcccc ctgaccagag cccacctgac cgaggtggaa
      841 agccggctgg aaagactgga acagctgttt ctgctgatct tcccacgcga ggacctggac
      901 atgatcctga agatggacag cctgcaggac atcaaggccc tgctgaccgg cctgttcgtg
      961 caggacaacg tgaacaagga cgccgtgacc gacagactgg ccagcgtgga aaccgacatg
     1021 cccctgaccc tgcggcagca cagaatcagc gccaccagca gcagcgagga aagcagcaac
     1081 aagggccagc ggcagctgac agtgtctgct gctgcaggcg gaagcggagg ctctggcgga
     1141 tctgagggcc agagcgacga gagagccctg ctggaccagc tgcacaccct gctgagcaac
     1201 accgacgcca ccggcctgga ggagatcgac agagccctgg gcatccccga gctggtgaac
     1261 cagggccagg ccctggagcc caagcaggac ggaggctggg gatctatggc cgaggaattc
     1321 gtgaccctga aggacgtggg catggacttc accctgggcg actgggagca gctgggcctg
     1381 gagcagggcg acaccttctg ggacaccgcc ctggacaact gccaggacct gttcctgctg
     1441 gacccccccg gcggaggaga cggccttggt ttgggacacg agaagttccc cagcgacctg
     1501 gacctggaca tgttcaacgg cagcctggag tgcgacatgg agagcatcat cagaagcgag
     1561 ctgatggacg ccgacggcct ggacttcaac ttcgacagct aggccgctcg aggagggccc
     1621 gaacaaaaac tcatctcaga agaggatctg aatagcgccg tcgaccatca tcataatcat
     1681 cattgagttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca gccatctgtt
     1741 gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac tgtcctttcc
     1801 taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat tctggggggt
     1861 ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca tgctggggat
     1921 gcggtgggct ctatggagat ctatcgatgt tctggggatg cggtgggctc tatggcttct
     1981 gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca
     2041 ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta
     2101 gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt
     2161 caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac
     2221 cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt ccgaagttcc
     2281 tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac cgcgacgtct
     2341 gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca gctctcggag
     2401 ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt cctgcgggta
     2461 aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt tgcatcggcc
     2521 gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct gacctattgc
     2581 atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga actgcccgct
     2641 gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct tagccagacg
     2701 agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg gcgtgatttc
     2761 atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga cgacaccgtc
     2821 agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga ctgccccgaa
     2881 gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga caatggccgc
     2941 ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata cgaggtcgcc
     3001 aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg ctacttcgag
     3061 cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct ccgcattggt
     3121 cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc ttgggcgcag
     3181 ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac acaaatcgcc
     3241 cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga tagtggaaac
     3301 cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga tttcgattcc
     3361 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     3421 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     3481 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     3541 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     3601 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     3661 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     3721 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     3781 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     3841 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     3901 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     3961 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     4021 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     4081 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     4141 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     4201 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     4261 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     4321 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     4381 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     4441 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     4501 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     4561 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     4621 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     4681 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     4741 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     4801 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     4861 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     4921 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     4981 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     5041 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     5101 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     5161 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     5221 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     5281 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     5341 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     5401 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     5461 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     5521 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     5581 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     5641 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     5701 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     5761 cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct cccgatcccc
     5821 tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag tatctgctcc
     5881 ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct acaacaaggc
     5941 aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt gcgctgcttc
     6001 gtacgggcca gatatacgcg ttgacattga ttattgacta gttattaata gtaatcaatt
     6061 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact ta
//
