LOCUS       sequence-468378-        6352 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the SCN1A-140 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 6352)
  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 6352)
  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: JPZF7 CMV-TO-SCN1A-140-NZF
FEATURES             Location/Qualifiers
     source          1..6352
                     /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             1954..1983
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(2039..2056)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2045..2269
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2441..2460)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2592..2639
                     /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             2647..3669
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2647..3669
                     /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    3799..3920
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3836..3855)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3890..3909
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3969..3985)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3969..3985)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3982..4004)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3993..4009
                     /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(4017..4023,4024..4041,4042..4047))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4062..4083
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4200..4217)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4371..4959)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4451..4470)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5130..5921,5922..5990))
                     /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     5753..5772
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5991..6095)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6170..6189)
                     /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 tgtataaatg caaggagtgt
      781 ggcaaggcct ttaagcagta cagtaatctg cctcagcaca agagaacaca caccggagaa
      841 cggccttacg attgtaggga atgcggaaaa gctttcagtc accgttcctc tctgagccgc
      901 cacttgatgt cccacactgg tgaaagaccc tatgcttgcg gtgaatgtgg tgaagctttc
      961 gcctggcttt cccaccttat ggagcatcac tctagtcaca ccggggaaag accgttcgtc
     1021 tgcggggtct gtaagatggg ctttagcctc ctgaccagcc tggcacagca tcacagttca
     1081 catacaggcg agcgccctta tgagtgccag gagtgcgggg aaaggttcat ctgtgggagt
     1141 acactcaaat gccatgagtc cgtgcacact ggtgagagac ctttcatatg tgaagtgtgt
     1201 cacaagagct acactcagtt cagcaacctc tgtagacaca agcgaatgca cgccgactgt
     1261 acaggggagc ggccttacaa atgcaactat tgcggcagaa gttataagca gcagtcaacc
     1321 cttgaggagc ataaagaacg atgccacact ggggagcgcc cctggcagtg tcgcatatgc
     1381 gaagacatgt tcgattccca ggagtatgtt aaacaacact gtatgtccct tgcatctcat
     1441 gctgctgcag gcggaagcgg aggctctggc ggatctgagg gccagagcga cgagagagcc
     1501 ctgctggacc agctgcacac cctgctgagc aacaccgacg ccaccggcct ggaggagatc
     1561 gacagagccc tgggcatccc cgagctggtg aaccagggcc aggccctgga gcccaagcag
     1621 gacggaggct ggggatctat ggccgaggaa ttcgtgaccc tgaaggacgt gggcatggac
     1681 ttcaccctgg gcgactggga gcagctgggc ctggagcagg gcgacacctt ctgggacacc
     1741 gccctggaca actgccagga cctgttcctg ctggaccccc ccggcggagg agacggcctt
     1801 ggtttgggac acgagaagtt ccccagcgac ctggacctgg acatgttcaa cggcagcctg
     1861 gagtgcgaca tggagagcat catcagaagc gagctgatgg acgccgacgg cctggacttc
     1921 aacttcgaca gctaggccgc tcgaggaggg cccgaacaaa aactcatctc agaagaggat
     1981 ctgaatagcg ccgtcgacca tcatcataat catcattgag tttaaacccg ctgatcagcc
     2041 tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg
     2101 accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat
     2161 tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag
     2221 gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatgga gatctatcga
     2281 tgttctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag ctggggctct
     2341 agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg
     2401 cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct
     2461 tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg gctcccttta
     2521 gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta gggtgatggt
     2581 tcacgtacct agaagttcct attccgaagt tcctattctc tagaaagtat aggaacttcc
     2641 ttggccaaaa agcctgaact caccgcgacg tctgtcgaga agtttctgat cgaaaagttc
     2701 gacagcgtct ccgacctgat gcagctctcg gagggcgaag aatctcgtgc tttcagcttc
     2761 gatgtaggag ggcgtggata tgtcctgcgg gtaaatagct gcgccgatgg tttctacaaa
     2821 gatcgttatg tttatcggca ctttgcatcg gccgcgctcc cgattccgga agtgcttgac
     2881 attggggaat tcagcgagag cctgacctat tgcatctccc gccgtgcaca gggtgtcacg
     2941 ttgcaagacc tgcctgaaac cgaactgccc gctgttctgc agccggtcgc ggaggccatg
     3001 gatgcgatcg ctgcggccga tcttagccag acgagcgggt tcggcccatt cggaccgcaa
     3061 ggaatcggtc aatacactac atggcgtgat ttcatatgcg cgattgctga tccccatgtg
     3121 tatcactggc aaactgtgat ggacgacacc gtcagtgcgt ccgtcgcgca ggctctcgat
     3181 gagctgatgc tttgggccga ggactgcccc gaagtccggc acctcgtgca cgcggatttc
     3241 ggctccaaca atgtcctgac ggacaatggc cgcataacag cggtcattga ctggagcgag
     3301 gcgatgttcg gggattccca atacgaggtc gccaacatct tcttctggag gccgtggttg
     3361 gcttgtatgg agcagcagac gcgctacttc gagcggaggc atccggagct tgcaggatcg
     3421 ccgcggctcc gggcgtatat gctccgcatt ggtcttgacc aactctatca gagcttggtt
     3481 gacggcaatt tcgatgatgc agcttgggcg cagggtcgat gcgacgcaat cgtccgatcc
     3541 ggagccggga ctgtcgggcg tacacaaatc gcccgcagaa gcgcggccgt ctggaccgat
     3601 ggctgtgtag aagtactcgc cgatagtgga aaccgacgcc ccagcactcg tccgagggca
     3661 aaggaatagc acgtactacg agatttcgat tccaccgccg ccttctatga aaggttgggc
     3721 ttcggaatcg ttttccggga cgccggctgg atgatcctcc agcgcgggga tctcatgctg
     3781 gagttcttcg cccaccccaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     3841 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     3901 aaactcatca atgtatctta tcatgtctgt ataccgtcga cctctagcta gagcttggcg
     3961 taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac
     4021 atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca
     4081 ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat
     4141 taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc
     4201 tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca
     4261 aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca
     4321 aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg
     4381 ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg
     4441 acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt
     4501 ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt
     4561 tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc
     4621 tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt
     4681 gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt
     4741 agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc
     4801 tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa
     4861 agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt
     4921 tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct
     4981 acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta
     5041 tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa
     5101 agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc
     5161 tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact
     5221 acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc
     5281 tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt
     5341 ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta
     5401 agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg
     5461 tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt
     5521 acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc
     5581 agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt
     5641 actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc
     5701 tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc
     5761 gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa
     5821 ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac
     5881 tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa
     5941 aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt
     6001 tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa
     6061 tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct
     6121 gacgtcgacg gatcgggaga tctcccgatc ccctatggtg cactctcagt acaatctgct
     6181 ctgatgccgc atagttaagc cagtatctgc tccctgcttg tgtgttggag gtcgctgagt
     6241 agtgcgcgag caaaatttaa gctacaacaa ggcaaggctt gaccgacaat tgcatgaaga
     6301 atctgcttag ggttaggcgt tttgcgctgc ttcgtacggg ccagatatac gc
//
