LOCUS       sequence-468403-        6211 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Plasmid encoding the UtroUp zinc finger array 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 6211)
  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 6211)
  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: EW368 CMV-TO-UtroUpZF-deImmunLink-NZF(FLP-IN)
FEATURES             Location/Qualifiers
     source          1..6211
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        291..494
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     445..465
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    496..514
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    517..535
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     545..569
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             1720..1749
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1805..1822)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1811..2035
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2207..2226)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2358..2405
                     /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             2413..3435
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2413..3435
                     /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    3565..3686
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3602..3621)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3656..3675
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3735..3751)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3735..3751)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3748..3770)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3759..3775
                     /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(3783..3789,3790..3807,3808..3813))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3828..3849
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3966..3983)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4137..4725)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4217..4236)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4896..5687,5688..5756))
                     /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     5519..5538
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5757..5861)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5936..5955)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        join(6122..6211,1..290)
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 cggtaaatgg cccgcctggc tgaccgccca acgacccccg cccattgacg tcaataatga
       61 cgtatgttcc catagtaacg ccaataggga ctttccattg acgtcaatgg gtggagtatt
      121 tacggtaaac tgcccacttg gcagtacatc aagtgtatca tatgccaagt acgcccccta
      181 ttgacgtcaa tgacggtaaa tggcccgcct ggcattatgc ccagtacatg accttatggg
      241 actttcctac ttggcagtac atctacgtat tagtcatcgc tattaccatg gtgatgcggt
      301 tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt ccaagtctcc
      361 accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac tttccaaaat
      421 gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg tgggaggtct
      481 atataagcag agctctccct atcagtgata gagatctccc tatcagtgat agagatcgtc
      541 gacgagctcg tttagtgaac cgtcagatcg cctggagacg ccatccacgc tgttttgacc
      601 tccatagaag acaccgggac cgatccagcc tccggactct agcgtttaaa cttaagcttc
      661 gccaccatga tattagaccg gccctatgcg tgcccagtcg aatcttgcga tcgtcgcttc
      721 agcagaagtg ataacttagt cagacacata aggatccaca cgggtcaaaa accgttccaa
      781 tgcaggattt gtatgaggaa tttctcgcgg agtgaccacc tcacgacaca caaccgcact
      841 cacaccggag agaaaccatt tgcatgcgat atatgcggca ggaagttcgc tgatccggga
      901 catctagtcc gtcacaatcg gatacacacc ggggaaaagc cgtttgcgtg ccctgtggaa
      961 agctgcgatc gaagattctc gcgctccgac gaactaactc gccatattag aatacatacc
     1021 ggacagaagc cctttcagtg tcggatctgc atgcgtaact tttcgtcaag ggacgtactg
     1081 agaagacata acaggacaca taccggggaa aagccatttg cgtgcgacat atgtgggaga
     1141 aagttcgctt cccgggatgt acttaggagg cataatcgga ttcatctacg ccaaaatgac
     1201 ttggaagctg ctgcatggga gctaggaatg tctggcggat ctgagggcca gagcgacgag
     1261 agagccctgc tggaccagct gcacaccctg ctgagcaaca ccgacgccac cggcctggag
     1321 gagatcgaca gagccctggg catccccgag ctggtgaacc agggccaggc cctggagccc
     1381 aagcaggacg gaggctgggg atctatggcc gaggaattcg tgaccctgaa ggacgtgggc
     1441 atggacttca ccctgggcga ctgggagcag ctgggcctgg agcagggcga caccttctgg
     1501 gacaccgccc tggacaactg ccaggacctg ttcctgctgg acccccccgg cggaggagac
     1561 ggccttggtt tgggacacga gaagttcccc agcgacctgg acctggacat gttcaacggc
     1621 agcctggagt gcgacatgga gagcatcatc agaagcgagc tgatggacgc cgacggcctg
     1681 gacttcaact tcgacagcta ggccgctcga ggagggcccg aacaaaaact catctcagaa
     1741 gaggatctga atagcgccgt cgaccatcat cataatcatc attgagttta aacccgctga
     1801 tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct
     1861 tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca
     1921 tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag
     1981 ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggagatc
     2041 tatcgatgtt ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg
     2101 ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg
     2161 gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc
     2221 ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc
     2281 cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt
     2341 gatggttcac gtacctagaa gttcctattc cgaagttcct attctctaga aagtatagga
     2401 acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa
     2461 aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc
     2521 agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc
     2581 tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg
     2641 cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt
     2701 gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag
     2761 gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga
     2821 ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc
     2881 catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct
     2941 ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg
     3001 gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg
     3061 agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg
     3121 tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca
     3181 ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc
     3241 ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc
     3301 cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg
     3361 accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg
     3421 agggcaaagg aatagcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg
     3481 ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc
     3541 atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa
     3601 agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt
     3661 ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc
     3721 ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca
     3781 cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa
     3841 ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3901 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc
     3961 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
     4021 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
     4081 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
     4141 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
     4201 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
     4261 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
     4321 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
     4381 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
     4441 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
     4501 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
     4561 tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc
     4621 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
     4681 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     4741 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     4801 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     4861 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     4921 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     4981 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5041 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5101 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5161 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5221 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5281 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5341 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     5401 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     5461 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     5521 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     5581 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     5641 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     5701 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     5761 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     5821 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     5881 ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa
     5941 tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg
     6001 ctgagtagtg cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca
     6061 tgaagaatct gcttagggtt aggcgttttg cgctgcttcg tacgggccag atatacgcgt
     6121 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc
     6181 ccatatatgg agttccgcgt tacataactt a
//
