LOCUS       sequence-471374-        6349 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the SCN1A-372 zinc finger array attached by a GS 
            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 6349)
  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 6349)
  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: JPZF10 CMV-TO-SCN1A-372-NZF
FEATURES             Location/Qualifiers
     source          1..6349
                     /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             1951..1980
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2036..2053)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2042..2266
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2438..2457)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2589..2636
                     /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             2644..3666
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2644..3666
                     /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    3796..3917
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3833..3852)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3887..3906
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3966..3982)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3966..3982)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3979..4001)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3990..4006
                     /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(4014..4020,4021..4038,4039..4044))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4059..4080
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4197..4214)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4368..4956)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4448..4467)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5127..5918,5919..5987))
                     /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     5750..5769
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5988..6092)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6167..6186)
                     /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 tgcatgggtg ccacctgtgc
      781 ggcaaggcgt tcacacattg cagcgacctg aggaaacacg aaagaactca cactggggag
      841 cgtccttacg aatgtaagga atgcggaaag gccttcatat gtggctacca gctcaccctt
      901 catcttagaa ctcacaccgg tgagagacca tttatctgcg aggtttgtca caaaagttat
      961 acccaatttt ctaacctgtg ccgccacaaa cggatgcacg ctgattgtac aggagagcga
     1021 cccttcgaat gtagtaaatg tggcaaggct tgtacacgaa gatgcaactt gatccaacat
     1081 cagaaggtac atacagggga gcggcccttt atctgcgaag tgtgtcacaa atcatatacc
     1141 cagttttcca acttatgtag acataagagg atgcatgccg actgcactgg ggaaagaccc
     1201 tataattgtg aagaatgcgg caaagctttt aacagatgct ctcacctgac tcgacacaag
     1261 aaaattcata ccggcgagag acctttccaa tgcgctctgt gccagaagtc cttcacccag
     1321 ctggctcatc tccagaagca tcacctggtt cacacgggcg agcggccata cacttgccac
     1381 ctttgtagga aggcctttac gcagtgttct catttgcgac gtcacgagaa gacgcatgct
     1441 gctgcaggcg gaagcggagg ctctggcgga tctgagggcc agagcgacga gagagccctg
     1501 ctggaccagc tgcacaccct gctgagcaac accgacgcca ccggcctgga ggagatcgac
     1561 agagccctgg gcatccccga gctggtgaac cagggccagg ccctggagcc caagcaggac
     1621 ggaggctggg gatctatggc cgaggaattc gtgaccctga aggacgtggg catggacttc
     1681 accctgggcg actgggagca gctgggcctg gagcagggcg acaccttctg ggacaccgcc
     1741 ctggacaact gccaggacct gttcctgctg gacccccccg gcggaggaga cggccttggt
     1801 ttgggacacg agaagttccc cagcgacctg gacctggaca tgttcaacgg cagcctggag
     1861 tgcgacatgg agagcatcat cagaagcgag ctgatggacg ccgacggcct ggacttcaac
     1921 ttcgacagct aggccgctcg cggagggccc gaacaaaaac tcatctcaga agaggatctg
     1981 aatagcgccg tcgaccatca tcataatcat cattgagttt aaacccgctg atcagcctcg
     2041 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     2101 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     2161 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     2221 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggagat ctatcgatgt
     2281 tctggggatg cggtgggctc tatggcttct gaggcggaaa gaaccagctg gggctctagg
     2341 gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc
     2401 agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc
     2461 tttctcgcca cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg
     2521 ttccgattta gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
     2581 cgtacctaga agttcctatt ccgaagttcc tattctctag aaagtatagg aacttccttg
     2641 gccaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac
     2701 agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat
     2761 gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat
     2821 cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt
     2881 ggggaattca gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg
     2941 caagacctgc ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat
     3001 gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga
     3061 atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat
     3121 cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag
     3181 ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc
     3241 tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg
     3301 atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct
     3361 tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg
     3421 cggctccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac
     3481 ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga
     3541 gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc
     3601 tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag
     3661 gaatagcacg tactacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc
     3721 ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag
     3781 ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     3841 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     3901 ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa
     3961 tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata
     4021 cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta
     4081 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4141 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg
     4201 ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag
     4261 gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa
     4321 ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc
     4381 cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca
     4441 ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg
     4501 accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct
     4561 catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt
     4621 gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag
     4681 tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc
     4741 agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac
     4801 actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga
     4861 gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc
     4921 aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg
     4981 gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca
     5041 aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt
     5101 atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca
     5161 gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg
     5221 atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca
     5281 ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt
     5341 cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt
     5401 agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca
     5461 cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca
     5521 tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga
     5581 agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact
     5641 gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga
     5701 gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg
     5761 ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc
     5821 tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga
     5881 tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat
     5941 gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt
     6001 caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt
     6061 atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac
     6121 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtaca atctgctctg
     6181 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt
     6241 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgc atgaagaatc
     6301 tgcttagggt taggcgtttt gcgctgcttc gtacgggcca gatatacgc
//
