LOCUS       sequence-468410-        6343 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Express mutated SCN1A-14 zinc finger array (G2E, A104K, S109I) 
            attached by a deimmunized linker to the NZF transcriptional 
            activation domain, 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 6343)
  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 6343)
  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: JPZF1 CMV-TO-SCN1A-377(G2E, A104K,
            S109I)-deIm...
FEATURES             Location/Qualifiers
     source          1..6343
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        4..383
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        384..587
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     538..558
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    589..607
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    610..628
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     638..662
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1945..1974
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2030..2047)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2036..2260
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2432..2451)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2583..2630
                     /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             2638..3660
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2638..3660
                     /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    3790..3911
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3827..3846)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3881..3900
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3960..3976)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3960..3976)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3973..3995)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3984..4000
                     /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(4008..4014,4015..4032,4033..4038))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4053..4074
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4191..4208)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4362..4950)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4442..4461)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5121..5912,5913..5981))
                     /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     5744..5763
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5982..6086)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6161..6180)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gttgacattg attattgact agttattaat agtaatcaat tacggggtca ttagttcata
       61 gcccatatat ggagttccgc gttacataac ttacggtaaa tggcccgcct ggctgaccgc
      121 ccaacgaccc ccgcccattg acgtcaataa tgacgtatgt tcccatagta acgccaatag
      181 ggactttcca ttgacgtcaa tgggtggagt atttacggta aactgcccac ttggcagtac
      241 atcaagtgta tcatatgcca agtacgcccc ctattgacgt caatgacggt aaatggcccg
      301 cctggcatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg
      361 tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat
      421 agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt
      481 tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc
      541 aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctctc cctatcagtg
      601 atagagatct ccctatcagt gatagagatc gtcgacgagc tcgtttagtg aaccgtcaga
      661 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      721 gcctccggac tctagcgttt aaacttaagc ttcgccacca tgtatgaatg caagaaatgt
      781 gggcggcgtt tcggccggct ttccaattgt acacgtcacg agaaaaccca cagcgcttgt
      841 acgggagagc gcccgcatgg ctgtcacttg tgtggtaaag cctttacaca ctgctcagat
      901 ttgagaaaac acgaaaggac acatacaggt gagcggccgt actcatgtgg aatatgtggg
      961 aaatcctttt ctgatagcag cgcaaagaga cggcactgta tcctgcacac gggagaacgg
     1021 cctttcgtct gtggggtctg caaaatggga ttttctctgc ttacgagctt aaagcagcat
     1081 cactccatac ataccgggga acgaccatat gagtgcaaag agtgtggaaa gacctttagg
     1141 caatgtagtc acctgaaaag gcatcaaagg atacatactg gcgagcggcc ctaccaatgt
     1201 gggcaatgtg gtaagagctt cagacaatct tccaatctgc atcagcatca ccgcctgcat
     1261 cacaccggtg aacgccctta tgaatgtaag gaatgcggaa agacgttcag acaatgttca
     1321 catctcaagc ggcatcaacg catccatacg ggagagaggc cgttcgagtg tgccgtctgc
     1381 ggagaatctt ttgtcaatcc ggccgaactc gcagaccacg ttaccgtgca tgctgctatg
     1441 ggcatgagcg gaggctctga cggatctgag ggccagagcg acgagagagc cctgctggac
     1501 cagctgcaca ccctgctgag caacaccgac gccaccggcc tggaggagat cgacagagcc
     1561 ctgggcatcc ccgagctggt gaaccagggc caggccctgg agcccaagca ggacggaggc
     1621 tggggatcta tggccgagga attcgtgacc ctgaaggacg tgggcatgga cttcaccctg
     1681 ggcgactggg agcagctggg cctggagcag ggcgacacct tctgggacac cgccctggac
     1741 aactgccagg acctgttcct gctggacccc cccggcggag gagacggcct tggtttggga
     1801 cacgagaagt tccccagcga cctggacctg gacatgttca acggcagcct ggagtgcgac
     1861 atggagagca tcatcagaag cgagctgatg gacgccgacg gcctggactt caacttcgac
     1921 agctaggccg ctcgaggagg gcccgaacaa aaactcatct cagaagagga tctgaatagc
     1981 gccgtcgacc atcatcataa tcatcattga gtttaaaccc gctgatcagc ctcgactgtg
     2041 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2101 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     2161 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     2221 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg agatctatcg atgttctggg
     2281 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     2341 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2401 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2461 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2521 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc
     2581 tagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc cttggccaaa
     2641 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc
     2701 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     2761 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     2821 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
     2881 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     2941 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc
     3001 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     3061 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     3121 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     3181 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     3241 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     3301 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     3361 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     3421 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     3481 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     3541 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     3601 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag
     3661 cacgtactac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     3721 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     3781 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3841 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3901 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     3961 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4021 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4081 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4141 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     4201 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4261 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4321 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4381 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4441 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4501 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4561 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4621 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4681 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4741 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4801 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4861 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4921 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4981 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5041 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5101 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5161 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5221 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     5281 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5341 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5401 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5461 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5521 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5581 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5641 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5701 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5761 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5821 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5881 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5941 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6001 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6061 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     6121 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
     6181 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
     6241 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
     6301 gggttaggcg ttttgcgctg cttcgtacgg gccagatata cgc
//
