LOCUS       sequence-468376-        5792 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter encoding GFP under control of SFFV promoter 
            with four ZNF35/ZNF250 binding sites upstream.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5792)
  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 5792)
  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: EW420 4x(ZNF35/ZNF250tarv3)-SFFV-GFP
FEATURES             Location/Qualifiers
     source          1..5792
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        319..726
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      734..743
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(749..751,752..1462)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             1490..1519
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1575..1592)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1581..1805
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1977..1996)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2128..2175
                     /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             2183..3205
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2183..3205
                     /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    3335..3456
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3372..3391)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3426..3445
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3505..3521)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3505..3521)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3518..3540)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3529..3545
                     /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(3553..3559,3560..3577,3578..3583))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3598..3619
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3736..3753)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3907..4495)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3987..4006)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4666..5457,5458..5526))
                     /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     5289..5308
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5527..5631)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5706..5725)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttaagctaca acaaggcaag gcttgaccga caattgacga ttgagtaaca ctgtatctag
       61 tctacctaca gggctcaaaa ttgcaaggat atataaagag ctcttcaaaa ttgcaaggat
      121 atataaagag gaggtcaaaa ttgcaaggat atataaagag cagaacaaaa ttgcaaggat
      181 atataaagag gccttccgta tcagtctaga gggtatataa tggatcccaa attgccgata
      241 aaataaaaga ttttatttag tctccagaaa aaggggggaa tgaaagaccc cacctgtagg
      301 tttggcaagc tagctgcagt aacgccattt tgcaaggcat ggaaaaatac caaaccaaga
      361 atagagaagt tcagatcaag ggcgggtaca tgaaaatagc taacgttggg ccaaacagga
      421 tatctgcggt gagcagtttc ggccccggcc cggggccaag aacagatggt caccgcagtt
      481 tcggccccgg cccgaggcca agaacagatg gtccccagat atggcccaac cctcagcagt
      541 ttcttaagac ccatcagatg tttccaggct cccccaagga cctgaaatga ccctgcgcct
      601 tatttgaatt aaccaatcag cctgcttctc gcttctgttc gcgcgcttct gcttcccgag
      661 ctctataaaa gagctcacaa cccctcactc ggcgcgccag tcctccgaca gactgagtcg
      721 cccgggaagc ttcgccacca tgggatccgt ctcaaagggc gaggaacttt ttacgggcgt
      781 tgttccgatc ctcgtcgaat tggacgggga cgttaatggg cataaattct cagttagtgg
      841 tgagggtgag ggggacgcga catatgggaa acttacgctg aagttcatat gtaccaccgg
      901 aaaattgcca gtgccgtggc ctactctcgt caccaccctg acatatggtg ttcagtgttt
      961 tagccggtat ccagaccata tgaaacagca cgattttttt aaatccgcta tgcctgaagg
     1021 ttacgtacag gagcgaacga tattttttaa agacgacgga aactacaaaa ctagagccga
     1081 ggtaaaattc gagggtgaca ctctcgtgaa ccggatcgaa ctgaagggca tagattttaa
     1141 ggaggatggc aatatactgg gccacaagct cgaatacaac tataattctc ataatgtcta
     1201 tatcatggcg gacaaacaga aaaacggcat caaggtaaac tttaaaatcc gacacaatat
     1261 tgaggacggc tctgtccagc ttgcggacca ttaccaacag aacacgccga taggggatgg
     1321 tccagtactt ctccctgata atcactattt gtctactcag tcagctctta gcaaggaccc
     1381 aaacgaaaaa cgggatcaca tggtccttct tgagttcgtc actgcggctg ggataacact
     1441 cgggatggac gaactgtata agaccggtta ggccgctcga ggagggcccg aacaaaaact
     1501 catctcagaa gaggatctga atagcgccgt cgaccatcat cataatcatc attgagttta
     1561 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     1621 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     1681 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     1741 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     1801 tatggagatc tatcgatgtt ctggggatgc ggtgggctct atggcttctg aggcggaaag
     1861 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     1921 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     1981 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2041 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2101 tgattagggt gatggttcac gtacctagaa gttcctattc cgaagttcct attctctaga
     2161 aagtatagga acttccttgg ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt
     2221 tctgatcgaa aagttcgaca gcgtctccga cctgatgcag ctctcggagg gcgaagaatc
     2281 tcgtgctttc agcttcgatg taggagggcg tggatatgtc ctgcgggtaa atagctgcgc
     2341 cgatggtttc tacaaagatc gttatgttta tcggcacttt gcatcggccg cgctcccgat
     2401 tccggaagtg cttgacattg gggaattcag cgagagcctg acctattgca tctcccgccg
     2461 tgcacagggt gtcacgttgc aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc
     2521 ggtcgcggag gccatggatg cgatcgctgc ggccgatctt agccagacga gcgggttcgg
     2581 cccattcgga ccgcaaggaa tcggtcaata cactacatgg cgtgatttca tatgcgcgat
     2641 tgctgatccc catgtgtatc actggcaaac tgtgatggac gacaccgtca gtgcgtccgt
     2701 cgcgcaggct ctcgatgagc tgatgctttg ggccgaggac tgccccgaag tccggcacct
     2761 cgtgcacgcg gatttcggct ccaacaatgt cctgacggac aatggccgca taacagcggt
     2821 cattgactgg agcgaggcga tgttcgggga ttcccaatac gaggtcgcca acatcttctt
     2881 ctggaggccg tggttggctt gtatggagca gcagacgcgc tacttcgagc ggaggcatcc
     2941 ggagcttgca ggatcgccgc ggctccgggc gtatatgctc cgcattggtc ttgaccaact
     3001 ctatcagagc ttggttgacg gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga
     3061 cgcaatcgtc cgatccggag ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc
     3121 ggccgtctgg accgatggct gtgtagaagt actcgccgat agtggaaacc gacgccccag
     3181 cactcgtccg agggcaaagg aatagcacgt actacgagat ttcgattcca ccgccgcctt
     3241 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     3301 cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag cttataatgg
     3361 ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt cactgcattc
     3421 tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac cgtcgacctc
     3481 tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     3541 cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg gtgcctaatg
     3601 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     3661 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     3721 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     3781 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     3841 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     3901 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     3961 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     4021 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     4081 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     4141 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     4201 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     4261 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     4321 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     4381 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     4441 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     4501 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     4561 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     4621 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     4681 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     4741 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     4801 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     4861 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     4921 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     4981 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     5041 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     5101 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     5161 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     5221 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     5281 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     5341 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     5401 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     5461 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     5521 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     5581 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     5641 ccgaaaagtg ccacctgacg tcgacggatc gggagatctc ccgatcccct atggtgcact
     5701 ctcagtacaa tctgctctga tgccgcatag ttaagccagt atctgctccc tgcttgtgtg
     5761 ttggaggtcg ctgagtagtg cgcgagcaaa at
//
