LOCUS       sequence-481140-        7421 bp    DNA     circular SYN 29-JUL-2025
DEFINITION  Cell fusion assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7421)
  AUTHORS   Brukman NG, Nakajima KP, Valansi C, Flyak K, Li X, Higashiyama T, 
            Podbilewicz B
  TITLE     A novel function for the sperm adhesion protein IZUMO1 in cell-cell 
            fusion.
  JOURNAL   J Cell Biol. 2023 Feb 6;222(2):e202207147. doi: 
            10.1083/jcb.202207147. Epub 2022 Nov 17.
  PUBMED    36394541
REFERENCE   2  (bases 1 to 7421)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jul 29, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "J Cell 
            Biol."; date: "2023-02-6"; volume: "222(2):e202207147. doi"; pages: 
            " 10.1083/jcb.202207147. Epub 2022 Nov 17"
COMMENT     Sequence Label: pCI_JUNO_H2B-RFP
FEATURES             Location/Qualifiers
     source          1..7421
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          662..1678
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1602..1624
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1686..1705
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     misc_feature    join(2656..3230,3231..3233,3234..3242)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2823..2840)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3050..3069
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3249..3626
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      3639..3648
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             3645..4322
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             3645..4322
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     primer_bind     complement(3775..3793)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4043..4062
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4237..4260
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(4413..4432)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4478..4533
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4532..4551)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4924..4940)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4924..4940)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4937..4959)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4948..4964
                     /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(4972..4978,4979..4996,4997..5002))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5017..5038
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5097..5292
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5143..5278
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5298..5432
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5335..5354)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5389..5408
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5500..5517)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5671..6259)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5751..5770)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6430..7221,7222..7290))
                     /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     7053..7072
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7291..7395)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat
       61 agcccatata tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg
      121 cccaacgacc cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
      181 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
      241 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
      301 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
      361 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
      421 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
      481 gcgatggggg cggggggggg gggggggsgc gccmgggggg gggggggggg gggggggggg
      541 ggggggggsg rggcggagag gtgcggcggc agccaatcag agcggcgcgc tccgaaagtt
      601 tccttttatg gcgaggcggc ggcggcggcg gccctataaa aagcgaagcg cgcggcgggc
      661 gggagtcgct gcgcgctgcc ttcgccccgt gccccgctcc gccgccgcct cgcgccgccc
      721 gccccggctc tgactgaccg cgttactccc acaggtgagc gggcgggacg gcccttctcc
      781 tccgggctgt aattagcgct tggtttaatg acggcttgtt tcttttctgt ggctgcgtga
      841 aagccttgag gggctccggg agggcccttt gtgcgggggg agcggctcgg ggggtgcgtg
      901 cgtgtgtgtg tgcgtgggga gcgccgcgtg cggctccgcg ctgcccggcg gctgtgagcg
      961 ctgcgggcgc ggcgcggggc tttgtgcgct ccgcagtgtg cgcgagggga gcgcggccgg
     1021 gggcggtgcc ccgcggtgcg gggggggctg cgaggggaac aaaggctgcg tgcggggtgt
     1081 gtgcgtgggg gggtgagcag ggggtgtggg cgcgtcggtc gggctgcaac cccccctgca
     1141 cccccctccc cgagttgctg agcacggccc ggcttcgggt gcggggctcc gtacggggcg
     1201 tggcgcgggg ctcgccgtgc cgggcggggg gtggcggcag gtgggggtgc cgggcggggc
     1261 ggggccgccy cgggccgggg rgggctcggg ggrggggcgc ggcggccccc ggagcgccgg
     1321 cggctgtcga ggcgcggcga gccgcagcca ttgcctttta tggtaatcgt gcgagagggc
     1381 gcagggactt cctttgtccc aaatctgtgc ggagccgaaa tctgggaggc gccgccgcac
     1441 cccctctagc gggcgcgggg cgaagcggtg cggcgccggc aggaaggaaa tgggcgggga
     1501 gggccttcgt gcgtcgccgc gccgccgtcc ccttctccct ctccagcctc ggggctgtcc
     1561 gcggggggac ggctgccttc gggggggacg gggcagggcg gggttcggct tctggcgtgt
     1621 gaccggcggc tctagagcct ctgctaacca tgttcatgcc ttcttctttt tcctacagct
     1681 cctgggcaac gtgctggtta ttgtgctgtc tcatcatttt ggcaaagaat tgctcgagct
     1741 ctttggcatc aggaggagca acaggagggc cctgccttga aggtcatggc acagtggtgg
     1801 cagatcctct tggggttgtg ggcagtccta cccaccttgg caggggacaa actgctcagc
     1861 gtctgcatga attccaagcg ccacaagcaa gaacctggcc cagaagacga actctaccag
     1921 gagtgcaggc cttgggagga caatgcctgc tgcacacgtt ccacaagttg ggaagcccac
     1981 cttgaggagc ccttgctctt taacttcagc atgatgcact gtggactgct gaccccggcc
     2041 tgtcgcaagc acttcatcca ggccatctgc ttccatgagt gttcccccaa cctggggcct
     2101 tggatccagc cggtggtccc aaacgggcag gaagagcagc gggtttgggg cgtgccgctg
     2161 tgccaggagg attgtgagga ctggtggcga gcctgccact catctttgac ttgtaaatcc
     2221 aactggctcc atggctggga ctggagtgag gagaagaaac actgccccgc acatgagccc
     2281 tgcctccctt tttcgtatca cttccccacc ccggatgacc tgtgtgagaa gatctggaac
     2341 aacacattca aggccagtcc cgagcgcagg aacagtgggc ggtgtctgca gaagtggttt
     2401 gagcccaccc ttagcaaccc caacgtggaa gtggccctcc actttgccgg ctctgcctta
     2461 gccccacagc tctcttacac cctcccagcc ttctctctgt gcctgttgtt ccatccctga
     2521 tccctgcttc cctctccaac ccacatttcc tgatccttct gggccatggc catctccatc
     2581 ctcaggctat tcatgttggg ggcagctagc tcatcgataa gcttgatatc gaattcctgc
     2641 agcccggggg atccgcccct ctccctcccc cccccctaac gttactggcc gaagccgctt
     2701 ggaataaggc cggtgtgcgt ttgtctatat gttattttcc accatattgc cgtcttttgg
     2761 caatgtgagg gcccggaaac ctggccctgt cttcttgacg agcattccta ggggtctttc
     2821 ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg aaggaagcag ttcctctgga
     2881 agcttcttga agacaaacaa cgtctgtagc gaccctttgc aggcagcgga accccccacc
     2941 tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa gatacacctg caaaggcggc
     3001 acaaccccag tgccacgttg tgagttggat agttgtggaa agagtcaaat ggctctcctc
     3061 aagcgtattc aacaaggggc tgaaggatgc ccagaaggta ccccattgta tgggatctga
     3121 tctggggcct cggtacacat gctttacatg tgtttagtcg aggttaaaaa aacgtctagg
     3181 ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat atggccacaa
     3241 ccatggttat gccagagcca gcgaagtctg ctcccgcccc gaaaaagggc tccaagaagg
     3301 cggtgactaa ggcgcagaag aaaggcggca agaagcgcaa gcgcagccgc aaggagagct
     3361 attccatcta tgtgtacaag gttctgaagc aggtccaccc tgacaccggc atttcgtcca
     3421 aggccatggg catcatgaat tcgtttgtga acgacatttt cgagcgcatc gcaggtgagg
     3481 cttcccgcct ggcgcattac aacaagcgct cgaccatcac ctccagggag atccagacgg
     3541 ccgtgcgcct gctgctgcct ggggagttgg ccaagcacgc cgtgtccgag ggtactaagg
     3601 ccatcaccaa gtacaccagc gctaaggatc caccggtcgc caccatggcc tcctccgagg
     3661 acgtcatcaa ggagttcatg cgcttcaagg tgcgcatgga gggctccgtg aacggccacg
     3721 agttcgagat cgagggcgag ggcgagggcc gcccctacga gggcacccag accgccaagc
     3781 tgaaggtgac caagggcggc cccctgccct tcgcctggga catcctgtcc cctcagttcc
     3841 agtacggctc caaggcctac gtgaagcacc ccgccgacat ccccgactac ttgaagctgt
     3901 ccttccccga gggcttcaag tgggagcgcg tgatgaactt cgaggacggc ggcgtggtga
     3961 ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat ctacaaggtg aagctgcgcg
     4021 gcaccaactt cccctccgac ggccccgtaa tgcagaagaa gaccatgggc tgggaggcct
     4081 ccaccgagcg gatgtacccc gaggacggcg ccctgaaggg cgagatcaag atgaggctga
     4141 agctgaagga cggcggccac tacgacgccg aggtcaagac cacctacatg gccaagaagc
     4201 ccgtgcagct gcccggcgcc tacaagaccg acatcaagct ggacatcacc tcccacaacg
     4261 aggactacac catcgtggaa cagtacgagc gcgccgaggg ccgccactcc accggcgcct
     4321 aagcggccgc aattcactcc tcaggtgcag gctgcctatc agaaggtggt ggctggtgtg
     4381 gccaatgccc tggctcacaa ataccactga gatctttttc cctctgccaa aaattatggg
     4441 gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt tattttcatt
     4501 gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg gagggcaaat
     4561 catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc ccatatgctg
     4621 gctgccatga acaaaggttg gctataaaga ggtcatcagt atatgaaaca gccccctgct
     4681 gtccattcct tattccatag aaaagccttg acttgaggtt agattttttt tatattttgt
     4741 tttgtgttat ttttttcttt aacatcccta aaattttcct tacatgtttt actagccaga
     4801 tttttcctcc tctcctgact actcccagtc atagctgtcc ctcttctctt atggagatcc
     4861 ctcgacctgc accgtcgacc agctggtcga cggtgcaccg tcgaccagct tggcgtaatc
     4921 atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg
     4981 agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat
     5041 tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc ggatccgcat
     5101 ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc cctaactccg
     5161 cccagttccg cccattctcc gccccatggc tgactaattt tttttattta tgcagaggcc
     5221 gaggccgcct cggcctctga gctattccag aagtagtgag gaggcttttt tggaggccta
     5281 ggcttttgca aaaagctaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5341 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5401 aactcatcaa tgtatcttat catgtctgga tccgctgcat taatgaatcg gccaacgcgc
     5461 ggggagaggc ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg
     5521 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5581 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5641 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5701 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5761 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5821 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5881 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5941 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     6001 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     6061 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     6121 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     6181 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     6241 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6301 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6361 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6421 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6481 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6541 atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc
     6601 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6661 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6721 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6781 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6841 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6901 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6961 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     7021 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     7081 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     7141 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     7201 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     7261 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7321 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7381 aataggggtt ccgcgcacat ttccccgaaa agtgccacct g
//
