LOCUS       sequence-481725-        6838 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Constitutive expression of H2B-eGFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6838)
  AUTHORS   Krueger D, Spoelstra WK, Mastebroek DJ, Kok RNU, Wu S, Nikolaev M, 
            Bannier-Helaouet M, Gjorevski N, Lutolf M, van Es J, van Zon J, Tans
            SJ, Clevers H
  TITLE     Epithelial tension controls intestinal cell extrusion.
  JOURNAL   Science. 2025 Sep 4;389(6764):eadr8753. doi: 
            10.1126/science.adr8753. Epub 2025 Sep 4.
  PUBMED    40906841
REFERENCE   2  (bases 1 to 6838)
  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: "Science."; 
            date: "2025-09-4"; volume: "389(6764):eadr8753. doi"; pages: " 
            10.1126/science.adr8753. Epub 2025 Sep 4"
COMMENT     Sequence Label: p2T-CMV-H2B-eGFP-SV40-Hygro
FEATURES             Location/Qualifiers
     source          1..6838
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(80..283)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(109..129)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        284..663
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     misc_feature    join(739..926,927..938)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(995..1015)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(995..1013)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1034..1050)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1034..1050)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1047..1069)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1058..1074
                     /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(1082..1088,1089..1106,1107..1112))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1127..1148
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1265..1282)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1436..2024)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1516..1535)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2195..2986,2987..3055))
                     /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     2818..2837
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3056..3160)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3186..3641)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3323..3344)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3535..3554
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3768..3790
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3782..3799
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3783..3799
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     promoter        3809..3827
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_feature    join(3858..3869,3870..4032)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     regulatory      4057..4066
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             complement(4074..5099)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVHRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(5108..5437)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5122..5257
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5480..5704
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     5693..5710
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(join(5735..6445,6446..6448,6449..6451))
                     /codon_start=1
                     /product="enhanced GFP with monomerizing A206K mutation 
                     (Zacharias et al., 2002)"
                     /label=mEGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSKLSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     primer_bind     complement(5777..5798)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     6126..6145
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     6385..6406
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     CDS             complement(join(6464..6838,1..3))
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKDGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAVTKYTSAK"
ORIGIN
        1 catggtgcta gcggatctca tagtgagtcg tattaatttc gataagccag taagcagtgg
       61 gttctctagt tagccagaga gctctgctta tatagacctc ccaccgtaca cgcctaccgc
      121 ccatttgcgt caatggggcg gagttgttac gacattttgg aaagtcccgt tgattttggt
      181 gccaaaacaa actcccattg acgtcaatgg ggtggagact tggaaatccc cgtgagtcaa
      241 accgctatcc acgcccattg atgtactgcc aaaaccgcat caccatggta atagcgatga
      301 ctaatacgta gatgtactgc caagtaggaa agtcccataa ggtcatgtac tgggcataat
      361 gccaggcggg ccatttaccg tcattgacgt caataggggg cgtacttggc atatgataca
      421 cttgatgtac tgccaagtgg gcagtttacc gtaaatactc cacccattga cgtcaatgga
      481 aagtccctat tggcgttact atgggaacat acgtcattat tgacgtcaat gggcgggggt
      541 cgttgggcgg tcagccaggc gggccattta ccgtaagtta tgtaacgcgg aactccatat
      601 atgggctatg aactaatgac cccgtaattg attactatta ataactagtc aataatcaat
      661 gtcaacgcgt atatctttac cgtaagttat gtaacgaaag caagtaacac cctaactgac
      721 acacattcca cagaattcaa gtgatctcca aaaaataagt actttttgac tgtaaataaa
      781 attgtaagga gtaaaaagta cttttttttc taaaaaaatg taattaagta aaagtaaaag
      841 tattgatttt taattgtact caagtaaagt aaaaatcccc aaaaataata cttaagtaca
      901 gtaatcaagt aaaattactc aagtacttta cacctctggt tcttgacccc ctaccttcag
      961 caagcccagc agatccgagc tccagctttt gttcccttta gtgagggtta attgcgcgct
     1021 tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     1081 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     1141 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     1201 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg
     1261 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     1321 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     1381 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     1441 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     1501 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     1561 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     1621 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     1681 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     1741 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     1801 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     1861 acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     1921 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     1981 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     2041 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     2101 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     2161 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     2221 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     2281 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     2341 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     2401 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     2461 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     2521 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     2581 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     2641 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     2701 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     2761 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     2821 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     2881 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     2941 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     3001 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     3061 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     3121 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     3181 cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     3241 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     3301 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     3361 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     3421 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     3481 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     3541 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     3601 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct
     3661 gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa
     3721 agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg
     3781 ttgtaaaacg acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac
     3841 cggcatatgg ttcttgacag aggtgtaaaa agtactcaaa aattttactc aagtgaaagt
     3901 acaagtactt agggaaaatt ttactcaatt aaaagtaaaa gtatctggct agaatcttac
     3961 ttgagtaaaa gtaaaaaagt actccattaa aattgtactt gagtattaag gaagtaaaag
     4021 taaaagcaag aaagatcgat ctcgaaggat ctggaggcca ccatggtgtc gatttattcc
     4081 tttgccctcg gacgagtgct ggggcgtcgg tttccactat cggcgagtac ttctacacag
     4141 ccatcggtcc agacggccgc gcttctgcgg gcgatttgtg tacgcccgac agtcccggct
     4201 ccggatcgga cgattgcgtc gcatcgaccc tgcgcccaag ctgcatcatc gaaattgccg
     4261 tcaaccaagc tctgatagag ttggtcaaga ccaatgcgga gcatatacgc ccggagccgc
     4321 ggcgatcctg caagctccgg atgcctccgc tcgaagtagc gcgtctgctg ctccatacaa
     4381 gccaaccacg gcctccagaa gaagatgttg gcgacctcgt attgggaatc cccgaacatc
     4441 gcctcgctcc agtcaatgac cgctgttatg cggccattgt ccgtcaggac attgttggag
     4501 ccgaaatccg cgtgcacgag gtgccggact tcggggcagt cctcggccca aagcatcagc
     4561 tcatcgagag cctgcgcgac ggacgcactg acggtgtcgt ccatcacagt ttgccagtga
     4621 tacacatggg gatcagcaat cgcgcatatg aaatcacgcc acgtagtgta ttgaccgatt
     4681 ccttgcggtc cgaatgggcc gaacccgctc gtctggctaa gatcggccgc agcgatcgca
     4741 tccatggcct ccgcgaccgg ctgcagaaca gcgggcagtt cggtttcagg caggtcttgc
     4801 aacgtgacac cctgtgcacg gcgggagatg caataggtca ggctctcgct gaattcccca
     4861 atgtcaagca cttccggaat cgggagcgcg gccgatgcaa agtgccgatg aacataacga
     4921 tctttgtaga aaccatcggc gcagctattt acccgcagga catatccacg ccctcctaca
     4981 tcgaagctga aagcacgaga ttcttcgccc tccgagagct gcatcaggtc ggagacgctg
     5041 tcgaactttt cgatcagaaa cttctcgaca gacgtcgcgg tgagttcagg ctttttcatg
     5101 gtcgcgtttt gcaaaagcct aggcctccaa aaaagcctcc tcactacttc tggaatagct
     5161 cagaggccga ggcggcctcg gcctctgcat aaataaaaaa aattagtcag ccatggggcg
     5221 gagaatgggc ggaactgggc ggagttaggg gcgggatggg cggagttagg ggcgggacta
     5281 tggttgctga ctaattgaga tgcatgcttt gcatacttct gcctgctggg gagcctgggg
     5341 actttccaca cctggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg
     5401 gggagcctgg ggactttcca caccctaact gacacacatt cgcgcgttta cccggggctg
     5461 gttctttccg cctcagaagc catagagccc accgcatccc cagcatgcct gctattgtct
     5521 tcccaatcct cccccttgct gtcctgcccc accccacccc ccagaataga atgacaccta
     5581 ctcagacaat gcgatgcaat ttcctcattt tattaggaaa ggacagtggg agtggcacct
     5641 tccagggtca aggaaggcac gggggagggg caaacaacag atggctggca actagaaggc
     5701 acagtcgagg ctgatcagcg ggtttaaacc cgctttagta cagctcgtcc atgccgagag
     5761 tgatcccggc ggcggtcacg aactccagca ggaccatgtg atcgcgcttc tcgttggggt
     5821 ctttgctcag cttggactgg gtgctcaggt agtggttgtc gggcagcagc acggggccgt
     5881 cgccgatggg ggtgttctgc tggtagtggt cggcgagctg cacgctgccg tcctcgatgt
     5941 tgtggcggat cttgaagttc accttgatgc cgttcttctg cttgtcggcc atgatataga
     6001 cgttgtggct gttgtagttg tactccagct tgtgccccag gatgttgccg tcctccttga
     6061 agtcgatgcc cttcagctcg atgcggttca ccagggtgtc gccctcgaac ttcacctcgg
     6121 cgcgggtctt gtagttgccg tcgtccttga agaagatggt gcgctcctgg acgtagcctt
     6181 cgggcatggc ggacttgaag aagtcgtgct gcttcatgtg gtcggggtag cggctgaagc
     6241 actgcacgcc gtaggtcagg gtggtcacga gggtgggcca gggcacgggc agcttgccgg
     6301 tggtgcagat gaacttcagg gtcagcttgc cgtaggtggc atcgccctcg ccctcgccgg
     6361 acacgctgaa cttgtggccg tttacgtcgc cgtccagctc gaccaggatg ggcaccaccc
     6421 cggtgaacag ctcctcgccc ttgctcacca tgaccggtgg atccttagcg ctggtgtact
     6481 tggtgacggc cttagtaccc tcggacacgg cgtgcttggc caactcccca ggcagcagca
     6541 ggcgcacggc cgtctggatc tccctggagg tgatggtcga gcgcttgttg taatgcgcca
     6601 ggcgggaagc ctcacctgcg atgcgctcga aaatgtcgtt cacaaacgaa ttcatgatgc
     6661 ccatggcctt ggacgaaatg ccggtgtcag ggtggacctg cttcagaacc ttgtacacat
     6721 agatggaata gctctccttg cggctgcgct tgcgcttctt gccgtctttc ttctgcgcct
     6781 tagtcaccgc cttcttggag ccctttttcg gggcgggagc agacttcgct ggctctgg
//
