LOCUS       sequence-468384-        7959 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Fluorescent reporter encoding H2B-GFP fusion under control of E1b 
            minimal promoter with the SCN1a promoter region encompassing 335 to 
            406 bp upstream of its transcriptional start site.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7959)
  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 7959)
  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: SCN1Aprom(335-406)-H2B-GFP
FEATURES             Location/Qualifiers
     source          1..7959
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     5'UTR           414..456
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     CDS             505..882
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     CDS             join(901..903,904..1614)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1642..1659)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    1648..1855
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(1888..1908)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        1893..2222
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2073..2208
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     2135..2154
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             2271..3296
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     CDS             2271..3296
                     /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(3356..3376)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    3418..3539
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3455..3474)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3509..3528
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     repeat_region   4302..4336
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4827..4843)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4827..4843)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4840..4862)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4851..4867
                     /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(4875..4881,4882..4899,4900..4905))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4920..4941
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5058..5075)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5229..5817)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5309..5328)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5988..6779,6780..6848))
                     /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     6611..6630
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6849..6953)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(6979..7434)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7116..7137)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7328..7347
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     7561..7583
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     7575..7592
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7576..7592
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   7722..7784
                     /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 ttcctctcag agacaattcc ttttcccctc atccttgtta aatgagctat
      301 cctttgcctt tatacgcaca gtctagaggg tatataatgg atcccatcgc gtctcagcct
      361 cactttgagc tcctccacac gaattccccg ataccgtcga ttcaaggagc ttgcttgttc
      421 tttttgcaga agctcagaat aaacgctcaa ctttggcaga tctaccaagc ttggtaccga
      481 gctcggatcc aactcgaggc caccatgcca gagccagcga agtctgctcc cgccccgaaa
      541 aagggctcca agaaggcggt gactaaggcg cagaagaaag gcggcaagaa gcgcaagcgc
      601 agccgcaagg agagctattc catctatgtg tacaaggttc tgaagcaggt ccaccctgac
      661 accggcattt cgtccaaggc catgggcatc atgaattcgt ttgtgaacga cattttcgag
      721 cgcatcgcag gtgaggcttc ccgcctggcg cattacaaca agcgctcgac catcacctcc
      781 agggagatcc agacggccgt gcgcctgctg ctgcctgggg agttggccaa gcacgccgtg
      841 tccgagggta ctaaggccat caccaagtac accagcgcta aggatccccg ggtaccggtc
      901 gtctcaaagg gcgaggaact ttttacgggc gttgttccga tcctcgtcga attggacggg
      961 gacgttaatg ggcataaatt ctcagttagt ggtgagggtg agggggacgc gacatatggg
     1021 aaacttacgc tgaagttcat atgtaccacc ggaaaattgc cggtgccgtg gcctactctc
     1081 gtcaccaccc tgacatatgg tgttcagtgt tttagccggt atccagacca tatgaaacag
     1141 cacgattttt ttaaatccgc tatgcctgaa ggttacgtac aggagcgaac gatatttttt
     1201 aaagacgacg gaaactacaa aactagagcc gaggtaaaat tcgagggtga cactctcgtg
     1261 aaccggatcg aactgaaggg catagatttt aaggaggatg gcaatatact gggccacaag
     1321 ctcgaataca actataattc tcataatgtc tatatcatgg cggacaaaca gaaaaacggc
     1381 atcaaggtaa actttaaaat ccgacacaat attgaggacg gctctgtcca gcttgcggac
     1441 cattaccaac agaacacgcc gataggggat ggtccagtac ttctccctga taatcactat
     1501 ttgtctactc agtcagctct tagcaaggac ccaaacgaaa aacgggatca catggtcctt
     1561 cttgagttcg tcactgcggc tgggataaca ctcgggatgg acgaactgta taagtagcta
     1621 gcgaattcga atttaaatcg gcctcgactg tgccttctag ttgccagcca tctgttgttt
     1681 gcccctcccc cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaat
     1741 aaaatgagga aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtgggg
     1801 tggggcagga cagcaagggg gaggattggg aagacaatag caggcatgct ggggacgcgg
     1861 ccgcaattta acgcgaatta attctgtgga atgtgtgtca gttagggtgt ggaaagtccc
     1921 caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt
     1981 gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt
     2041 cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg
     2101 cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct
     2161 ctgcctctga gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca
     2221 aaaagctccc gggagcttgt atatccattt tcggatctga tcagcacgtg atgaaaaagc
     2281 ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg
     2341 acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc
     2401 gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt
     2461 atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca
     2521 gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc
     2581 ctgaaaccga actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg
     2641 cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat
     2701 acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa
     2761 ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt
     2821 gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg
     2881 tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg
     2941 attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc
     3001 agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg
     3061 cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg
     3121 atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg
     3181 tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag
     3241 tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaataggtcg
     3301 acaatcaacc tctggattac aaaatttgtg aaagattgac tggtattctt aactatgttg
     3361 ctccttttac gctatgtgga tacgctgctt taatgccttt gtatcatgcg ttaactaaac
     3421 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     3481 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat
     3541 catgtctgga attgactcaa atgatgtcaa ttagtctatc agaagctcat ctggtctccc
     3601 ttccggggga caagacatcc ctgtttaata tttaaacagc agtgttccca aactgggttc
     3661 ttatatccct tgctctggtc aaccaggttg cagggtttcc tgtcctcaca ggaacgaagt
     3721 ccctaaagaa acagtggcag ccaggtttag ccccggaatt gactggattc cttttttagg
     3781 gcccattggt atggcttttt ccccgtatcc ccccaggtgt ctgcaggctc aaagagcagc
     3841 gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc cgtgcccggg ctgtccccgc
     3901 acgctgccgg ctcggggatg cggggggagc gccggaccgg agcggagccc cgggcggctc
     3961 gctgctgccc cctagcgggg gagggacgta attacatccc tgggggcttt gggggggggc
     4021 tgtccctgat atctataaca agaaaatata tatataataa gttatcacgt aagtagaaca
     4081 tgaaataaca atataattat cgtatgagtt aaatcttaaa agtcacgtaa aagataatca
     4141 tgcgtcattt tgactcacgc ggtcgttata gttcaaaatc agtgacactt accgcattga
     4201 caagcacgcc tcacgggagc tccaagcggc gactgagatg tcctaaatgc acagcgacgg
     4261 attcgcgcta tttagaaaga gagagcaata tttcaagaat gcatgcgtca attttacgca
     4321 gactatcttt ctagggttaa tctagctgca tcaggatcat atcgtcgggt cttttttccg
     4381 gctcagtcat cgcccaagct ggcgctatct gggcatcggg gaggaagaag cccgtgcctt
     4441 ttcccgcgag gttgaagcgg catggaaaga gtttgccgag gatgactgct gctgcattga
     4501 cgttgagcga aaacgcacgt ttaccatgat gattcgggaa ggtgtggcca tgcacgcctt
     4561 taacggtgaa ctgttcgttc aggccacctg ggataccagt tcgtcgcggc ttttccggac
     4621 acagttccgg atggtcagcc cgaagcgcat cagcaacccg aacaataccg gcgacagccg
     4681 gaactgccgt gccggtgtgc agattaatga cagcggtgcg gcgctgggat attacgtcag
     4741 cgaggacggg tatcctggct ggatgccgca gaaatggaca tggatacccc gtgagttacc
     4801 cggcgggcgc gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg
     4861 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa
     4921 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4981 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     5041 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
     5101 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca
     5161 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg
     5221 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt
     5281 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc
     5341 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct
     5401 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc
     5461 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta
     5521 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca
     5581 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag
     5641 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag
     5701 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt
     5761 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
     5821 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg
     5881 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga
     5941 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta
     6001 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc
     6061 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg
     6121 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga
     6181 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt
     6241 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt
     6301 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc
     6361 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc
     6421 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca
     6481 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
     6541 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg
     6601 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa
     6661 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa
     6721 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga
     6781 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga
     6841 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
     6901 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt
     6961 ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt
     7021 tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat
     7081 caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat
     7141 taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac
     7201 tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc
     7261 ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga
     7321 gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca
     7381 cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc gcgtcccatt
     7441 cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct tcgctattac
     7501 gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg ccagggtttt
     7561 cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc ctcgttcatt cacgtttttg
     7621 aacccgtgga ggacgggcag actcgcggtg caaatgtgtt ttacagcgtg atggagcaga
     7681 tgaagatgct cgacacgctg cagaacacgc agctagatta accctagaaa gataatcata
     7741 ttgtgacgta cgttaaagat aatcatgtgt aaaattgacg catgtgtttt atcggtctgt
     7801 atatcgaggt ttatttatta atttgaatag atattaagtt ttattatatt tacacttaca
     7861 tactaataat aaattcaaca aacaatttat ttatgtttat ttatttatta aaaaaaacaa
     7921 aaactcaaaa tttcttctat aaagtaacaa aacttttat
//
