LOCUS       sequence-468466-        6040 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the UTRN-14 zinc finger array attached by a GS 
            linker to the VP64 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 6040)
  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 6040)
  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: EW172 CMV-TO-UTRN-14ZF-VP64 (FLP-IN)
FEATURES             Location/Qualifiers
     source          1..6040
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        7..386
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        387..590
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     541..561
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    592..610
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    613..631
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     641..665
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1468..1617
                     /codon_start=1
                     /product="tetrameric repeat of the minimal activation 
                     domain of herpes simplex virus VP16 (Beerli et al., 1998)"
                     /label=VP64
                     /translation="DALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDF
                     DLDML"
     CDS             1645..1674
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1730..1747)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1736..1960
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2132..2151)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2283..2330
                     /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             2338..3360
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2338..3360
                     /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    3490..3611
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3527..3546)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3581..3600
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3660..3676)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3660..3676)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3673..3695)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3684..3700
                     /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(3708..3714,3715..3732,3733..3738))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3753..3774
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3891..3908)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4062..4650)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4142..4161)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4821..5612,5613..5681))
                     /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     5444..5463
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5682..5786)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5861..5880)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 cgcgttgaca ttgattattg actagttatt aatagtaatc aattacgggg tcattagttc
       61 atagcccata tatggagttc cgcgttacat aacttacggt aaatggcccg cctggctgac
      121 cgcccaacga cccccgccca ttgacgtcaa taatgacgta tgttcccata gtaacgccaa
      181 tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc cacttggcag
      241 tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac ggtaaatggc
      301 ccgcctggca ttatgcccag tacatgacct tatgggactt tcctacttgg cagtacatct
      361 acgtattagt catcgctatt accatggtga tgcggttttg gcagtacatc aatgggcgtg
      421 gatagcggtt tgactcacgg ggatttccaa gtctccaccc cattgacgtc aatgggagtt
      481 tgttttggca ccaaaatcaa cgggactttc caaaatgtcg taacaactcc gccccattga
      541 cgcaaatggg cggtaggcgt gtacggtggg aggtctatat aagcagagct ctccctatca
      601 gtgatagaga tctccctatc agtgatagag atcgtcgacg agctcgttta gtgaaccgtc
      661 agatcgcctg gagacgccat ccacgctgtt ttgacctcca tagaagacac cgggaccgat
      721 ccagcctccg gactctagcg tttaaactta agcttcgcca ccatgcacga gtgtgcccga
      781 tgtgggaaga atttctcttg gcactcagac ctcatcttgc acgagcaaat ccacacagga
      841 gagcgacctt tcaagtgtta caagtgtgga cgtggattcg cagagcacgg gacactcaat
      901 cgacacctta gaactaaggg cggatgtact ggggagcggc cttacaattg tgaggagtgt
      961 ggaagagctt tctcccaagc tagccatctc caagaccacc aaaggcttca cactggggag
     1021 agaccccacc aatgtatcga gtgtggaaag tccttcaata gacactgtaa tcttataagg
     1081 caccaaaaga tacacacggg tgagcggcca ttcgagtgtt ctaagtgtgg gaaggcttgt
     1141 actagaaggt gtaatttgat acaacaccaa aaggtccaca ctggtgagcg tcctttcgct
     1201 tgtaggttct gtgctaagcc tttcagacgt tcttcagaca tgcgagacca cgagagagtc
     1261 cacactgggg agagaccatt cgcatgtcgg ttctgtgcca agcctttcag acgatcttcc
     1321 gacatgcggg accacgagag ggttcacaca ggggagcgtc ccttccaatg tgagctctgt
     1381 tcatacactt gtcccagaag gtcaaattta gacagacaca tgaagtccca cgctgctgca
     1441 ggcggaagcg gaggctctgg cggatctgat gccctggacg acttcgacct ggatatgctg
     1501 ggcagcgacg ccctggatga ttttgatctg gacatgctgg gatctgacgc tctggacgat
     1561 ttcgatctcg acatgttggg atcagatgca ctggatgact ttgacctgga catgctcgga
     1621 tcataggccg ctcgaggagg gcccgaacaa aaactcatct cagaagagga tctgaatagc
     1681 gccgtcgacc atcatcataa tcatcattga gtttaaaccc gctgatcagc ctcgactgtg
     1741 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     1801 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     1861 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     1921 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg agatctatcg atgttctggg
     1981 gatgcggtgg gctctatggc ttctgaggcg gaaagaacca gctggggctc tagggggtat
     2041 ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg
     2101 accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc
     2161 gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga
     2221 tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtacc
     2281 tagaagttcc tattccgaag ttcctattct ctagaaagta taggaacttc cttggccaaa
     2341 aagcctgaac tcaccgcgac gtctgtcgag aagtttctga tcgaaaagtt cgacagcgtc
     2401 tccgacctga tgcagctctc ggagggcgaa gaatctcgtg ctttcagctt cgatgtagga
     2461 gggcgtggat atgtcctgcg ggtaaatagc tgcgccgatg gtttctacaa agatcgttat
     2521 gtttatcggc actttgcatc ggccgcgctc ccgattccgg aagtgcttga cattggggaa
     2581 ttcagcgaga gcctgaccta ttgcatctcc cgccgtgcac agggtgtcac gttgcaagac
     2641 ctgcctgaaa ccgaactgcc cgctgttctg cagccggtcg cggaggccat ggatgcgatc
     2701 gctgcggccg atcttagcca gacgagcggg ttcggcccat tcggaccgca aggaatcggt
     2761 caatacacta catggcgtga tttcatatgc gcgattgctg atccccatgt gtatcactgg
     2821 caaactgtga tggacgacac cgtcagtgcg tccgtcgcgc aggctctcga tgagctgatg
     2881 ctttgggccg aggactgccc cgaagtccgg cacctcgtgc acgcggattt cggctccaac
     2941 aatgtcctga cggacaatgg ccgcataaca gcggtcattg actggagcga ggcgatgttc
     3001 ggggattccc aatacgaggt cgccaacatc ttcttctgga ggccgtggtt ggcttgtatg
     3061 gagcagcaga cgcgctactt cgagcggagg catccggagc ttgcaggatc gccgcggctc
     3121 cgggcgtata tgctccgcat tggtcttgac caactctatc agagcttggt tgacggcaat
     3181 ttcgatgatg cagcttgggc gcagggtcga tgcgacgcaa tcgtccgatc cggagccggg
     3241 actgtcgggc gtacacaaat cgcccgcaga agcgcggccg tctggaccga tggctgtgta
     3301 gaagtactcg ccgatagtgg aaaccgacgc cccagcactc gtccgagggc aaaggaatag
     3361 cacgtactac gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc
     3421 gttttccggg acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc
     3481 gcccacccca acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     3541 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     3601 aatgtatctt atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg
     3661 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     3721 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     3781 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     3841 ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact
     3901 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     3961 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4021 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4081 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4141 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4201 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4261 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4321 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4381 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4441 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4501 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4561 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4621 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     4681 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     4741 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     4801 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     4861 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     4921 gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
     4981 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5041 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5101 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5161 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5221 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5281 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5341 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5401 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5461 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5521 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5581 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     5641 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     5701 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     5761 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtcgac
     5821 ggatcgggag atctcccgat cccctatggt gcactctcag tacaatctgc tctgatgccg
     5881 catagttaag ccagtatctg ctccctgctt gtgtgttgga ggtcgctgag tagtgcgcga
     5941 gcaaaattta agctacaaca aggcaaggct tgaccgacaa ttgcatgaag aatctgctta
     6001 gggttaggcg ttttgcgctg cttcgtacgg gccagatata
//
