LOCUS       sequence-468372-        8004 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fuorescent reporter encoding H2B-GFP fusion under control of E1b 
            minimal promoter with four ZNF35/ZNFIKZF1 binding sites upstream.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8004)
  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 8004)
  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: EW387 (ZNF35/IKZF1tar-6bp spacers)x4-H2B-GFP
FEATURES             Location/Qualifiers
     source          1..8004
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(join(169..960,961..1029))
                     /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     792..811
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1030..1134)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1160..1615)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1297..1318)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     1509..1528
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1742..1764
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     1756..1773
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     1757..1773
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   1903..1965
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     5'UTR           2599..2641
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             2690..3067
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             join(3086..3088,3089..3799)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3827..3844)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3833..4040
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4073..4093)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4078..4407
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4258..4393
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4320..4339
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4456..5481
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             4456..5481
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     primer_bind     complement(5541..5561)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    5603..5724
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5640..5659)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5694..5713
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   6487..6521
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(7012..7028)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7012..7028)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(7025..7047)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    7036..7052
                     /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(7060..7066,7067..7084,7085..7090))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7105..7126
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(7243..7260)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7414..8002)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7494..7513)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
       61 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
      121 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
      181 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
      241 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
      301 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
      361 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
      421 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
      481 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
      541 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
      601 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
      661 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
      721 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
      781 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
      841 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
      901 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
      961 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     1021 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     1081 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     1141 tccccgaaaa gtgccaccta aattgtaagc gttaatattt tgttaaaatt cgcgttaaat
     1201 ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa tcggcaaaat cccttataaa
     1261 tcaaaagaat agaccgagat agggttgagt gttgttccag tttggaacaa gagtccacta
     1321 ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg tctatcaggg cgatggccca
     1381 ctacgtgaac catcacccta atcaagtttt ttggggtcga ggtgccgtaa agcactaaat
     1441 cggaacccta aagggagccc ccgatttaga gcttgacggg gaaagccggc gaacgtggcg
     1501 agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg cgctggcaag tgtagcggtc
     1561 acgctgcgcg taaccaccac acccgccgcg cttaatgcgc cgctacaggg cgcgtcccat
     1621 tcgccattca ggctgcgcaa ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta
     1681 cgccagctgg cgaaaggggg atgtgctgca aggcgattaa gttgggtaac gccagggttt
     1741 tcccagtcac gacgttgtaa aacgacggcc agtgagcgcg cctcgttcat tcacgttttt
     1801 gaacccgtgg aggacgggca gactcgcggt gcaaatgtgt tttacagcgt gatggagcag
     1861 atgaagatgc tcgacacgct gcagaacacg cagctagatt aaccctagaa agataatcat
     1921 attgtgacgt acgttaaaga taatcatgtg taaaattgac gcatgtgttt tatcggtctg
     1981 tatatcgagg tttatttatt aatttgaata gatattaagt tttattatat ttacacttac
     2041 atactaataa taaattcaac aaacaattta tttatgttta tttatttatt aaaaaaaaca
     2101 aaaactcaaa atttcttcta taaagtaaca aaacttttat gagggacagc ccccccccaa
     2161 agcccccagg gatgtaatta cgtccctccc ccgctagggg gcagcagcga gccgcccggg
     2221 gctccgctcc ggtccggcgc tccccccgca tcccagagcc ggcagcgtgc ggggacagcc
     2281 cgggcacggg gaaggtggca cgggatcgct ttcctctgaa cgcttctcgc tgctctttga
     2341 gcctgcagac acctgggggg atacggggaa aaggcctcca cggccacaat tgacgattga
     2401 gtaaacagga aatatataaa gagctcttca aacaggaaat atataaagag gaggtcaaac
     2461 aggaaatata taaagagcag aacaaacagg aaatatataa agagagtcta gagggtatat
     2521 aatggatccc atcgcgtctc agcctcactt tgagctcctc cacacgaatt ccccgatacc
     2581 gtcgattcaa ggagcttgct tgttcttttt gcagaagctc agaataaacg ctcaactttg
     2641 gcagatctac caagcttggt accgagctcg gatccaactc gaggccacca tgccagagcc
     2701 agcgaagtct gctcccgccc cgaaaaaggg ctccaagaag gcggtgacta aggcgcagaa
     2761 gaaaggcggc aagaagcgca agcgcagccg caaggagagc tattccatct atgtgtacaa
     2821 ggttctgaag caggtccacc ctgacaccgg catttcgtcc aaggccatgg gcatcatgaa
     2881 ttcgtttgtg aacgacattt tcgagcgcat cgcaggtgag gcttcccgcc tggcgcatta
     2941 caacaagcgc tcgaccatca cctccaggga gatccagacg gccgtgcgcc tgctgctgcc
     3001 tggggagttg gccaagcacg ccgtgtccga gggtactaag gccatcacca agtacaccag
     3061 cgctaaggat ccccgggtac cggtcgtctc aaagggcgag gaacttttta cgggcgttgt
     3121 tccgatcctc gtcgaattgg acggggacgt taatgggcat aaattctcag ttagtggtga
     3181 gggtgagggg gacgcgacat atgggaaact tacgctgaag ttcatatgta ccaccggaaa
     3241 attgccggtg ccgtggccta ctctcgtcac caccctgaca tatggtgttc agtgttttag
     3301 ccggtatcca gaccatatga aacagcacga tttttttaaa tccgctatgc ctgaaggtta
     3361 cgtacaggag cgaacgatat tttttaaaga cgacggaaac tacaaaacta gagccgaggt
     3421 aaaattcgag ggtgacactc tcgtgaaccg gatcgaactg aagggcatag attttaagga
     3481 ggatggcaat atactgggcc acaagctcga atacaactat aattctcata atgtctatat
     3541 catggcggac aaacagaaaa acggcatcaa ggtaaacttt aaaatccgac acaatattga
     3601 ggacggctct gtccagcttg cggaccatta ccaacagaac acgccgatag gggatggtcc
     3661 agtacttctc cctgataatc actatttgtc tactcagtca gctcttagca aggacccaaa
     3721 cgaaaaacgg gatcacatgg tccttcttga gttcgtcact gcggctggga taacactcgg
     3781 gatggacgaa ctgtataagt agctagcgaa ttcgaattta aatcggcctc gactgtgcct
     3841 tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac cctggaaggt
     3901 gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg tctgagtagg
     3961 tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga ttgggaagac
     4021 aatagcaggc atgctgggga cgcggccgca atttaacgcg aattaattct gtggaatgtg
     4081 tgtcagttag ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     4141 catctcaatt agtcagcaac caggtgtgga aagtccccag gctccccagc aggcagaagt
     4201 atgcaaagca tgcatctcaa ttagtcagca accatagtcc cgcccctaac tccgcccatc
     4261 ccgcccctaa ctccgcccag ttccgcccat tctccgcccc atggctgact aatttttttt
     4321 atttatgcag aggccgaggc cgcctctgcc tctgagctat tccagaagta gtgaggaggc
     4381 ttttttggag gcctaggctt ttgcaaaaag ctcccgggag cttgtatatc cattttcgga
     4441 tctgatcagc acgtgatgaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
     4501 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
     4561 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
     4621 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
     4681 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
     4741 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
     4801 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
     4861 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
     4921 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
     4981 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
     5041 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
     5101 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
     5161 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
     5221 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
     5281 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
     5341 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
     5401 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
     5461 cgtccgaggg caaaggaata ggtcgacaat caacctctgg attacaaaat ttgtgaaaga
     5521 ttgactggta ttcttaacta tgttgctcct tttacgctat gtggatacgc tgctttaatg
     5581 cctttgtatc atgcgttaac taaacttgtt tattgcagct tataatggtt acaaataaag
     5641 caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta gttgtggttt
     5701 gtccaaactc atcaatgtat cttatcatgt ctggaattga ctcaaatgat gtcaattagt
     5761 ctatcagaag ctcatctggt ctcccttccg ggggacaaga catccctgtt taatatttaa
     5821 acagcagtgt tcccaaactg ggttcttata tcccttgctc tggtcaacca ggttgcaggg
     5881 tttcctgtcc tcacaggaac gaagtcccta aagaaacagt ggcagccagg tttagccccg
     5941 gaattgactg gattcctttt ttagggccca ttggtatggc tttttccccg tatcccccca
     6001 ggtgtctgca ggctcaaaga gcagcgagaa gcgttcagag gaaagcgatc ccgtgccacc
     6061 ttccccgtgc ccgggctgtc cccgcacgct gccggctcgg ggatgcgggg ggagcgccgg
     6121 accggagcgg agccccgggc ggctcgctgc tgccccctag cgggggaggg acgtaattac
     6181 atccctgggg gctttggggg ggggctgtcc ctgatatcta taacaagaaa atatatatat
     6241 aataagttat cacgtaagta gaacatgaaa taacaatata attatcgtat gagttaaatc
     6301 ttaaaagtca cgtaaaagat aatcatgcgt cattttgact cacgcggtcg ttatagttca
     6361 aaatcagtga cacttaccgc attgacaagc acgcctcacg ggagctccaa gcggcgactg
     6421 agatgtccta aatgcacagc gacggattcg cgctatttag aaagagagag caatatttca
     6481 agaatgcatg cgtcaatttt acgcagacta tctttctagg gttaatctag ctgcatcagg
     6541 atcatatcgt cgggtctttt ttccggctca gtcatcgccc aagctggcgc tatctgggca
     6601 tcggggagga agaagcccgt gccttttccc gcgaggttga agcggcatgg aaagagtttg
     6661 ccgaggatga ctgctgctgc attgacgttg agcgaaaacg cacgtttacc atgatgattc
     6721 gggaaggtgt ggccatgcac gcctttaacg gtgaactgtt cgttcaggcc acctgggata
     6781 ccagttcgtc gcggcttttc cggacacagt tccggatggt cagcccgaag cgcatcagca
     6841 acccgaacaa taccggcgac agccggaact gccgtgccgg tgtgcagatt aatgacagcg
     6901 gtgcggcgct gggatattac gtcagcgagg acgggtatcc tggctggatg ccgcagaaat
     6961 ggacatggat accccgtgag ttacccggcg ggcgcgcttg gcgtaatcat ggtcatagct
     7021 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     7081 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     7141 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     7201 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     7261 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     7321 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     7381 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     7441 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     7501 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     7561 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     7621 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     7681 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     7741 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     7801 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     7861 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     7921 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     7981 gcgcagaaaa aaaggatctc aaga
//
