LOCUS       sequence-468452-        5784 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter encoding GFP under control of SFFV promoter 
            with four ZNF35/ZNF35 binding sites upstream.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5784)
  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 5784)
  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: EW434 4x(ZNF35/ZNF35tar)-SFFV-GFP
FEATURES             Location/Qualifiers
     source          1..5784
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        311..718
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      726..735
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(741..743,744..1454)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             1482..1511
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1567..1584)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1573..1797
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1969..1988)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2120..2167
                     /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             2175..3197
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2175..3197
                     /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    3327..3448
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3364..3383)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3418..3437
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3497..3513)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3497..3513)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3510..3532)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3521..3537
                     /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(3545..3551,3552..3569,3570..3575))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3590..3611
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3728..3745)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3899..4487)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3979..3998)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4658..5449,5450..5518))
                     /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     5281..5300
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5519..5623)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5698..5717)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttaagctaca acaaggcaag gcttgaccga caattgacga ttgagtaaca ctgtatctag
       61 tctacctaca gggctcatat ataaagagat atataaagag ctcttcatat ataaagagat
      121 atataaagag gaggtcatat ataaagagat atataaagag cagaacatat ataaagagat
      181 atataaagag tatcagtcta gagggtatat aatggatccc aaattgccga taaaataaaa
      241 gattttattt agtctccaga aaaagggggg aatgaaagac cccacctgta ggtttggcaa
      301 gctagctgca gtaacgccat tttgcaaggc atggaaaaat accaaaccaa gaatagagaa
      361 gttcagatca agggcgggta catgaaaata gctaacgttg ggccaaacag gatatctgcg
      421 gtgagcagtt tcggccccgg cccggggcca agaacagatg gtcaccgcag tttcggcccc
      481 ggcccgaggc caagaacaga tggtccccag atatggccca accctcagca gtttcttaag
      541 acccatcaga tgtttccagg ctcccccaag gacctgaaat gaccctgcgc cttatttgaa
      601 ttaaccaatc agcctgcttc tcgcttctgt tcgcgcgctt ctgcttcccg agctctataa
      661 aagagctcac aacccctcac tcggcgcgcc agtcctccga cagactgagt cgcccgggaa
      721 gcttcgccac catgggatcc gtctcaaagg gcgaggaact ttttacgggc gttgttccga
      781 tcctcgtcga attggacggg gacgttaatg ggcataaatt ctcagttagt ggtgagggtg
      841 agggggacgc gacatatggg aaacttacgc tgaagttcat atgtaccacc ggaaaattgc
      901 cagtgccgtg gcctactctc gtcaccaccc tgacatatgg tgttcagtgt tttagccggt
      961 atccagacca tatgaaacag cacgattttt ttaaatccgc tatgcctgaa ggttacgtac
     1021 aggagcgaac gatatttttt aaagacgacg gaaactacaa aactagagcc gaggtaaaat
     1081 tcgagggtga cactctcgtg aaccggatcg aactgaaggg catagatttt aaggaggatg
     1141 gcaatatact gggccacaag ctcgaataca actataattc tcataatgtc tatatcatgg
     1201 cggacaaaca gaaaaacggc atcaaggtaa actttaaaat ccgacacaat attgaggacg
     1261 gctctgtcca gcttgcggac cattaccaac agaacacgcc gataggggat ggtccagtac
     1321 ttctccctga taatcactat ttgtctactc agtcagctct tagcaaggac ccaaacgaaa
     1381 aacgggatca catggtcctt cttgagttcg tcactgcggc tgggataaca ctcgggatgg
     1441 acgaactgta taagaccggt taggccgctc gaggagggcc cgaacaaaaa ctcatctcag
     1501 aagaggatct gaatagcgcc gtcgaccatc atcataatca tcattgagtt taaacccgct
     1561 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     1621 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     1681 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     1741 agggggagga ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggaga
     1801 tctatcgatg ttctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     1861 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     1921 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     1981 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     2041 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     2101 gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag
     2161 gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     2221 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     2281 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     2341 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     2401 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     2461 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     2521 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     2581 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     2641 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     2701 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     2761 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     2821 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     2881 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     2941 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     3001 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     3061 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     3121 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     3181 cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa
     3241 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     3301 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     3361 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     3421 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     3481 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     3541 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     3601 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3661 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3721 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3781 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3841 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3901 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3961 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     4021 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     4081 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     4141 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     4201 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     4261 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     4321 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     4381 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     4441 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     4501 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4561 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4621 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4681 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4741 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4801 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4861 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4921 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     4981 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     5041 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     5101 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     5161 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     5221 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     5281 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     5341 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     5401 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     5461 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     5521 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5581 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5641 tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca ctctcagtac
     5701 aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg tgttggaggt
     5761 cgctgagtag tgcgcgagca aaat
//
