LOCUS       sequence-468417-        6034 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the UTRN-43 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 6034)
  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 6034)
  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: EW171 CMV-TO-UTRN-43ZF-VP64 (FLP-IN)
FEATURES             Location/Qualifiers
     source          1..6034
                     /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             1459..1608
                     /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             1636..1665
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1721..1738)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1727..1951
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2123..2142)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2274..2321
                     /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             2329..3351
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2329..3351
                     /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    3481..3602
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3518..3537)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3572..3591
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3651..3667)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3651..3667)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3664..3686)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3675..3691
                     /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(3699..3705,3706..3723,3724..3729))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3744..3765
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3882..3899)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4053..4641)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4133..4152)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4812..5603,5604..5672))
                     /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     5435..5454
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5673..5777)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5852..5871)
                     /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 tgttcgagtg tgccgtctgt
      781 ggggagtcct tcgtaaatcc tgcagagttg gccgaccacg ttacagttca caccggggag
      841 agacccttcg agtgtgccgt ttgtggggag tctttcgtca atccagccga gctcgctgac
      901 cacgtcacag tccacactgg ggagcgacct tacgagtgtc aagagtgtca aaagacgttc
      961 tctaggtcat cccacttgct ccgtcaccaa agtgtccact gtactggaga gagacctttc
     1021 acgtgtgact catgtggttt cggtttctct tgtgagaagc tcctcgacga gcacgtcttg
     1081 acgtgtacgg gcgagcgtcc ttacgagtgt gggcaatgtg gacggtactt catccaaatg
     1141 gcagacttcc acaggcacga gaagtgtcac acaggggagc gaccatacga gtgtgaggac
     1201 tgtgggttgg gtttcgtaga cttaactgac ttaactgacc accaaaaggt ccacacaggg
     1261 gagagaccat tcgagtgtgc agtctgtgga gagtccttcg taaatcccgc cgagcttgca
     1321 gaccacgtta cagtccacac tggggagagg ccacaagagt gtccagtttg tcacaagatc
     1381 atacacggag ctggaaaact tccacgtcac atgaggacgc acgctgctgc aggcggaagc
     1441 ggaggctctg gcggatctga tgccctggac gacttcgacc tggatatgct gggcagcgac
     1501 gccctggatg attttgatct ggacatgctg ggatctgacg ctctggacga tttcgatctc
     1561 gacatgttgg gatcagatgc actggatgac tttgacctgg acatgctcgg atcataggcc
     1621 gctcgaggag ggcccgaaca aaaactcatc tcagaagagg atctgaatag cgccgtcgac
     1681 catcatcata atcatcattg agtttaaacc cgctgatcag cctcgactgt gccttctagt
     1741 tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga aggtgccact
     1801 cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag taggtgtcat
     1861 tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga agacaatagc
     1921 aggcatgctg gggatgcggt gggctctatg gagatctatc gatgttctgg ggatgcggtg
     1981 ggctctatgg cttctgaggc ggaaagaacc agctggggct ctagggggta tccccacgcg
     2041 ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca
     2101 cttgccagcg ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc
     2161 gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct
     2221 ttacggcacc tcgaccccaa aaaacttgat tagggtgatg gttcacgtac ctagaagttc
     2281 ctattccgaa gttcctattc tctagaaagt ataggaactt ccttggccaa aaagcctgaa
     2341 ctcaccgcga cgtctgtcga gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg
     2401 atgcagctct cggagggcga agaatctcgt gctttcagct tcgatgtagg agggcgtgga
     2461 tatgtcctgc gggtaaatag ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg
     2521 cactttgcat cggccgcgct cccgattccg gaagtgcttg acattgggga attcagcgag
     2581 agcctgacct attgcatctc ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa
     2641 accgaactgc ccgctgttct gcagccggtc gcggaggcca tggatgcgat cgctgcggcc
     2701 gatcttagcc agacgagcgg gttcggccca ttcggaccgc aaggaatcgg tcaatacact
     2761 acatggcgtg atttcatatg cgcgattgct gatccccatg tgtatcactg gcaaactgtg
     2821 atggacgaca ccgtcagtgc gtccgtcgcg caggctctcg atgagctgat gctttgggcc
     2881 gaggactgcc ccgaagtccg gcacctcgtg cacgcggatt tcggctccaa caatgtcctg
     2941 acggacaatg gccgcataac agcggtcatt gactggagcg aggcgatgtt cggggattcc
     3001 caatacgagg tcgccaacat cttcttctgg aggccgtggt tggcttgtat ggagcagcag
     3061 acgcgctact tcgagcggag gcatccggag cttgcaggat cgccgcggct ccgggcgtat
     3121 atgctccgca ttggtcttga ccaactctat cagagcttgg ttgacggcaa tttcgatgat
     3181 gcagcttggg cgcagggtcg atgcgacgca atcgtccgat ccggagccgg gactgtcggg
     3241 cgtacacaaa tcgcccgcag aagcgcggcc gtctggaccg atggctgtgt agaagtactc
     3301 gccgatagtg gaaaccgacg ccccagcact cgtccgaggg caaaggaata gcacgtacta
     3361 cgagatttcg attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg
     3421 gacgccggct ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc
     3481 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     3541 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     3601 tatcatgtct gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg
     3661 tttcctgtgt gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata
     3721 aagtgtaaag cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca
     3781 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3841 gcggggagag gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg
     3901 cgctcggtcg ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta
     3961 tccacagaat caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc
     4021 aggaaccgta aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag
     4081 catcacaaaa atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac
     4141 caggcgtttc cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc
     4201 ggatacctgt ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt
     4261 aggtatctca gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc
     4321 gttcagcccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga
     4381 cacgacttat cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta
     4441 ggcggtgcta cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta
     4501 tttggtatct gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga
     4561 tccggcaaac aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg
     4621 cgcagaaaaa aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag
     4681 tggaacgaaa actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc
     4741 tagatccttt taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact
     4801 tggtctgaca gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt
     4861 cgttcatcca tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta
     4921 ccatctggcc ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta
     4981 tcagcaataa accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc
     5041 gcctccatcc agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat
     5101 agtttgcgca acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt
     5161 atggcttcat tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg
     5221 tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca
     5281 gtgttatcac tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta
     5341 agatgctttt ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg
     5401 cgaccgagtt gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact
     5461 ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg
     5521 ctgttgagat ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt
     5581 actttcacca gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga
     5641 ataagggcga cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc
     5701 atttatcagg gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa
     5761 caaatagggg ttccgcgcac atttccccga aaagtgccac ctgacgtcga cggatcggga
     5821 gatctcccga tcccctatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa
     5881 gccagtatct gctccctgct tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaattt
     5941 aagctacaac aaggcaaggc ttgaccgaca attgcatgaa gaatctgctt agggttaggc
     6001 gttttgcgct gcttcgtacg ggccagatat acgc
//
