LOCUS       sequence-468422-        5738 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter encoding GFP under control of SFFV promoter 
            with four ZNF35/IKZF1 binding sites upstream.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5738)
  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 5738)
  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: EW435 4x(ZNF35/IKZF1tar)-SFFV-GFP
FEATURES             Location/Qualifiers
     source          1..5738
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        265..672
                     /label=SFFV promoter
                     /note="spleen focus-forming virus long terminal repeat 
                     (LTR) promoter"
     regulatory      680..689
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(695..697,698..1408)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             1436..1465
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     primer_bind     complement(1521..1538)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1527..1751
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1923..1942)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    2074..2121
                     /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             2129..3151
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             2129..3151
                     /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    3281..3402
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3318..3337)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3372..3391
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(3451..3467)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3451..3467)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3464..3486)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3475..3491
                     /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(3499..3505,3506..3523,3524..3529))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3544..3565
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3682..3699)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3853..4441)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3933..3952)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4612..5403,5404..5472))
                     /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     5235..5254
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5473..5577)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(5652..5671)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ttaagctaca acaaggcaag gcttgaccga caattgacga ttgagtaaac aggaaatata
       61 taaagagctc ttcaaacagg aaatatataa agaggaggtc aaacaggaaa tatataaaga
      121 gcagaacaaa caggaaatat ataaagagag tctagagggt atataatgga tcccaaattg
      181 ccgataaaat aaaagatttt atttagtctc cagaaaaagg ggggaatgaa agaccccacc
      241 tgtaggtttg gcaagctagc tgcagtaacg ccattttgca aggcatggaa aaataccaaa
      301 ccaagaatag agaagttcag atcaagggcg ggtacatgaa aatagctaac gttgggccaa
      361 acaggatatc tgcggtgagc agtttcggcc ccggcccggg gccaagaaca gatggtcacc
      421 gcagtttcgg ccccggcccg aggccaagaa cagatggtcc ccagatatgg cccaaccctc
      481 agcagtttct taagacccat cagatgtttc caggctcccc caaggacctg aaatgaccct
      541 gcgccttatt tgaattaacc aatcagcctg cttctcgctt ctgttcgcgc gcttctgctt
      601 cccgagctct ataaaagagc tcacaacccc tcactcggcg cgccagtcct ccgacagact
      661 gagtcgcccg ggaagcttcg ccaccatggg atccgtctca aagggcgagg aactttttac
      721 gggcgttgtt ccgatcctcg tcgaattgga cggggacgtt aatgggcata aattctcagt
      781 tagtggtgag ggtgaggggg acgcgacata tgggaaactt acgctgaagt tcatatgtac
      841 caccggaaaa ttgccagtgc cgtggcctac tctcgtcacc accctgacat atggtgttca
      901 gtgttttagc cggtatccag accatatgaa acagcacgat ttttttaaat ccgctatgcc
      961 tgaaggttac gtacaggagc gaacgatatt ttttaaagac gacggaaact acaaaactag
     1021 agccgaggta aaattcgagg gtgacactct cgtgaaccgg atcgaactga agggcataga
     1081 ttttaaggag gatggcaata tactgggcca caagctcgaa tacaactata attctcataa
     1141 tgtctatatc atggcggaca aacagaaaaa cggcatcaag gtaaacttta aaatccgaca
     1201 caatattgag gacggctctg tccagcttgc ggaccattac caacagaaca cgccgatagg
     1261 ggatggtcca gtacttctcc ctgataatca ctatttgtct actcagtcag ctcttagcaa
     1321 ggacccaaac gaaaaacggg atcacatggt ccttcttgag ttcgtcactg cggctgggat
     1381 aacactcggg atggacgaac tgtataagac cggttaggcc gctcgaggag ggcccgaaca
     1441 aaaactcatc tcagaagagg atctgaatag cgccgtcgac catcatcata atcatcattg
     1501 agtttaaacc cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg
     1561 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     1621 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     1681 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt
     1741 gggctctatg gagatctatc gatgttctgg ggatgcggtg ggctctatgg cttctgaggc
     1801 ggaaagaacc agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag
     1861 cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc
     1921 cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc
     1981 tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa
     2041 aaaacttgat tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc
     2101 tctagaaagt ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga
     2161 gaagtttctg atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga
     2221 agaatctcgt gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag
     2281 ctgcgccgat ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct
     2341 cccgattccg gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc
     2401 ccgccgtgca cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct
     2461 gcagccggtc gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg
     2521 gttcggccca ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg
     2581 cgcgattgct gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc
     2641 gtccgtcgcg caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg
     2701 gcacctcgtg cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac
     2761 agcggtcatt gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat
     2821 cttcttctgg aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag
     2881 gcatccggag cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga
     2941 ccaactctat cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg
     3001 atgcgacgca atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag
     3061 aagcgcggcc gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg
     3121 ccccagcact cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc
     3181 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     3241 ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta
     3301 taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact
     3361 gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc
     3421 gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     3481 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     3541 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     3601 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     3661 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     3721 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     3781 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3841 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     3901 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3961 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     4021 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4081 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4141 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4201 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4261 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4321 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4381 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4441 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4501 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4561 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4621 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     4681 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     4741 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     4801 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     4861 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     4921 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     4981 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     5041 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     5101 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5161 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5221 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5281 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5341 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5401 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5461 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5521 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5581 atttccccga aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg
     5641 tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct
     5701 tgtgtgttgg aggtcgctga gtagtgcgcg agcaaaat
//
