LOCUS       sequence-468443-        7958 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter encoding H2B-GFP fusion under control of E1b 
            minimal promoter with the UTRN promoter region encompassing 14 to 67
            bp upstream of its transcriptional start site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7958)
  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 7958)
  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: EW411 (UTRN-43-5bp spacer-UTRN-14)-H2B-GFP
FEATURES             Location/Qualifiers
     source          1..7958
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     5'UTR           413..455
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             504..881
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             join(900..902,903..1613)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1641..1658)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1647..1854
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1887..1907)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1892..2221
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2072..2207
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2134..2153
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2270..3295
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2270..3295
                     /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(3355..3375)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3417..3538
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3454..3473)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3508..3527
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4301..4335
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4826..4842)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4826..4842)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4839..4861)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4850..4866
                     /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(4874..4880,4881..4898,4899..4904))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4919..4940
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5057..5074)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5228..5816)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5308..5327)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5987..6778,6779..6847))
                     /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     6610..6629
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6848..6952)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6978..7433)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7115..7136)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7327..7346
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7560..7582
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7574..7591
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7575..7591
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   7721..7783
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 gagggacagc ccccccccaa agcccccagg gatgtaatta cgtccctccc ccgctagggg
       61 gcagcagcga gccgcccggg gctccgctcc ggtccggcgc tccccccgca tcccagagcc
      121 ggcagcgtgc ggggacagcc cgggcacggg gaaggtggca cgggatcgct ttcctctgaa
      181 cgcttctcgc tgctctttga gcctgcagac acctgggggg atacggggaa aaggcctcca
      241 cggccacaat tgacgattga gtggcaggcc cgcagcgggg aggaggagga ggagccgccg
      301 aaggagcgag cctctatcag tctagagggt atataatgga tcccatcgcg tctcagcctc
      361 actttgagct cctccacacg aattccccga taccgtcgat tcaaggagct tgcttgttct
      421 ttttgcagaa gctcagaata aacgctcaac tttggcagat ctaccaagct tggtaccgag
      481 ctcggatcca actcgaggcc accatgccag agccagcgaa gtctgctccc gccccgaaaa
      541 agggctccaa gaaggcggtg actaaggcgc agaagaaagg cggcaagaag cgcaagcgca
      601 gccgcaagga gagctattcc atctatgtgt acaaggttct gaagcaggtc caccctgaca
      661 ccggcatttc gtccaaggcc atgggcatca tgaattcgtt tgtgaacgac attttcgagc
      721 gcatcgcagg tgaggcttcc cgcctggcgc attacaacaa gcgctcgacc atcacctcca
      781 gggagatcca gacggccgtg cgcctgctgc tgcctgggga gttggccaag cacgccgtgt
      841 ccgagggtac taaggccatc accaagtaca ccagcgctaa ggatccccgg gtaccggtcg
      901 tctcaaaggg cgaggaactt tttacgggcg ttgttccgat cctcgtcgaa ttggacgggg
      961 acgttaatgg gcataaattc tcagttagtg gtgagggtga gggggacgcg acatatggga
     1021 aacttacgct gaagttcata tgtaccaccg gaaaattgcc agtgccgtgg cctactctcg
     1081 tcaccaccct gacatatggt gttcagtgtt ttagccggta tccagaccat atgaaacagc
     1141 acgatttttt taaatccgct atgcctgaag gttacgtaca ggagcgaacg atatttttta
     1201 aagacgacgg aaactacaaa actagagccg aggtaaaatt cgagggtgac actctcgtga
     1261 accggatcga actgaagggc atagatttta aggaggatgg caatatactg ggccacaagc
     1321 tcgaatacaa ctataattct cataatgtct atatcatggc ggacaaacag aaaaacggca
     1381 tcaaggtaaa ctttaaaatc cgacacaata ttgaggacgg ctctgtccag cttgcggacc
     1441 attaccaaca gaacacgccg ataggggatg gtccagtact tctccctgat aatcactatt
     1501 tgtctactca gtcagctctt agcaaggacc caaacgaaaa acgggatcac atggtccttc
     1561 ttgagttcgt cactgcggct gggataacac tcgggatgga cgaactgtat aagtagctag
     1621 cgaattcgaa tttaaatcgg cctcgactgt gccttctagt tgccagccat ctgttgtttg
     1681 cccctccccc gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata
     1741 aaatgaggaa attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt
     1801 ggggcaggac agcaaggggg aggattggga agacaatagc aggcatgctg gggacgcggc
     1861 cgcaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc
     1921 aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg
     1981 tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc
     2041 agcaaccata gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc
     2101 ccattctccg ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc
     2161 tgcctctgag ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa
     2221 aaagctcccg ggagcttgta tatccatttt cggatctgat cagcacgtga tgaaaaagcc
     2281 tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca gcgtctccga
     2341 cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg taggagggcg
     2401 tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc gttatgttta
     2461 tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg gggaattcag
     2521 cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc aagacctgcc
     2581 tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg cgatcgctgc
     2641 ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa tcggtcaata
     2701 cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc actggcaaac
     2761 tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc tgatgctttg
     2821 ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct ccaacaatgt
     2881 cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga tgttcgggga
     2941 ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt gtatggagca
     3001 gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc ggctccgggc
     3061 gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg gcaatttcga
     3121 tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag ccgggactgt
     3181 cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct gtgtagaagt
     3241 actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg aataggtcga
     3301 caatcaacct ctggattaca aaatttgtga aagattgact ggtattctta actatgttgc
     3361 tccttttacg ctatgtggat acgctgcttt aatgcctttg tatcatgcgt taactaaact
     3421 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     3481 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     3541 atgtctggaa ttgactcaaa tgatgtcaat tagtctatca gaagctcatc tggtctccct
     3601 tccgggggac aagacatccc tgtttaatat ttaaacagca gtgttcccaa actgggttct
     3661 tatatccctt gctctggtca accaggttgc agggtttcct gtcctcacag gaacgaagtc
     3721 cctaaagaaa cagtggcagc caggtttagc cccggaattg actggattcc ttttttaggg
     3781 cccattggta tggctttttc cccgtatccc cccaggtgtc tgcaggctca aagagcagcg
     3841 agaagcgttc agaggaaagc gatcccgtgc caccttcccc gtgcccgggc tgtccccgca
     3901 cgctgccggc tcggggatgc ggggggagcg ccggaccgga gcggagcccc gggcggctcg
     3961 ctgctgcccc ctagcggggg agggacgtaa ttacatccct gggggctttg ggggggggct
     4021 gtccctgata tctataacaa gaaaatatat atataataag ttatcacgta agtagaacat
     4081 gaaataacaa tataattatc gtatgagtta aatcttaaaa gtcacgtaaa agataatcat
     4141 gcgtcatttt gactcacgcg gtcgttatag ttcaaaatca gtgacactta ccgcattgac
     4201 aagcacgcct cacgggagct ccaagcggcg actgagatgt cctaaatgca cagcgacgga
     4261 ttcgcgctat ttagaaagag agagcaatat ttcaagaatg catgcgtcaa ttttacgcag
     4321 actatctttc tagggttaat ctagctgcat caggatcata tcgtcgggtc ttttttccgg
     4381 ctcagtcatc gcccaagctg gcgctatctg ggcatcgggg aggaagaagc ccgtgccttt
     4441 tcccgcgagg ttgaagcggc atggaaagag tttgccgagg atgactgctg ctgcattgac
     4501 gttgagcgaa aacgcacgtt taccatgatg attcgggaag gtgtggccat gcacgccttt
     4561 aacggtgaac tgttcgttca ggccacctgg gataccagtt cgtcgcggct tttccggaca
     4621 cagttccgga tggtcagccc gaagcgcatc agcaacccga acaataccgg cgacagccgg
     4681 aactgccgtg ccggtgtgca gattaatgac agcggtgcgg cgctgggata ttacgtcagc
     4741 gaggacgggt atcctggctg gatgccgcag aaatggacat ggataccccg tgagttaccc
     4801 ggcgggcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4861 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4921 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4981 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     5041 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     5101 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     5161 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     5221 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5281 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5341 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5401 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5461 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5521 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5581 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5641 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5701 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5761 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5821 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5881 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5941 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     6001 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     6061 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     6121 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     6181 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6241 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6301 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6361 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6421 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6481 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6541 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6601 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6661 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6721 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6781 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6841 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6901 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6961 cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg cgttaaattt
     7021 ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc
     7081 aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga gtccactatt
     7141 aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg atggcccact
     7201 acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag cactaaatcg
     7261 gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga acgtggcgag
     7321 aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg tagcggtcac
     7381 gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg cgtcccattc
     7441 gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt cgctattacg
     7501 ccagctggcg aaagggggat gtgctgcaag gcgattaagt tgggtaacgc cagggttttc
     7561 ccagtcacga cgttgtaaaa cgacggccag tgagcgcgcc tcgttcattc acgtttttga
     7621 acccgtggag gacgggcaga ctcgcggtgc aaatgtgttt tacagcgtga tggagcagat
     7681 gaagatgctc gacacgctgc agaacacgca gctagattaa ccctagaaag ataatcatat
     7741 tgtgacgtac gttaaagata atcatgtgta aaattgacgc atgtgtttta tcggtctgta
     7801 tatcgaggtt tatttattaa tttgaataga tattaagttt tattatattt acacttacat
     7861 actaataata aattcaacaa acaatttatt tatgtttatt tatttattaa aaaaaacaaa
     7921 aactcaaaat ttcttctata aagtaacaaa acttttat
//
