LOCUS       sequence-481141-        7930 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 7930)
  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 7930)
  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_IZUMO1-V5_H2B-RFP
FEATURES             Location/Qualifiers
     source          1..7930
                     /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"
     CDS             3078..3119
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3129..3146
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    join(3165..3739,3740..3742,3743..3751)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3332..3349)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3559..3578
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             3758..4135
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      4148..4157
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4154..4831
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             4154..4831
                     /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(4284..4302)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4552..4571
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     4746..4769
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(4922..4941)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4987..5042
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5041..5060)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5433..5449)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5433..5449)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5446..5468)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5457..5473
                     /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(5481..5487,5488..5505,5506..5511))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5526..5547
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5606..5801
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5652..5787
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5807..5941
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5844..5863)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5898..5917
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6009..6026)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6180..6768)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6260..6279)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6939..7730,7731..7799))
                     /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     7562..7581
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7800..7904)
                     /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 gggggsgcgc gccmggsggg gsggggsggg gggrgggggg
      541 ggggggggng nggcggagag 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 ggggccgcck 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 tgctcgagag
     1741 ttcgaagcct taagtacgcg taggcgcgcc ttaattaacg tttaaacgct agcgaattcg
     1801 gtcgactgga tccggtaccg aggagatctg ccgccgcgat cgccatgggg ccgcatttta
     1861 cactcttgct ggcagctctt gccaactgcc tgtgtccagg gaggccctgc atcaaatgtg
     1921 accagtttgt gacagatgcg ctaaagactt tcgaaaacac ttacctgaat gaccacctgc
     1981 cacacgacat tcacaaaaat gtaatgagga tggtgaacca tgaagtatcg agcttcggcg
     2041 tagtcacttc ggctgaggat tcctatttgg gggccgtgga cgagaacaca ctggaacaag
     2101 caacctggag ttttctgaag gatctgaagc gtattacaga cagtgactta aaaggagagc
     2161 tctttataaa ggaactattg tggatgcttc gtcatcaaaa ggacatcttt aacaatcttg
     2221 ctagacagtt ccaaaaggaa gttctttgtc ccaacaaatg cggagtgatg tcgcagactt
     2281 tgatctggtg tcttaagtgc gaaaagcagt tgcacatttg tcggaaatcc ctagattgtg
     2341 gagagcgcca catagaggta catcgctcgg aagacctggt cctggactgt ctgctcagtt
     2401 ggcatcgtgc ttctaaggga cttacagatt acagttttta cagggtttgg gagaacagtt
     2461 ctgagacctt gattgccaag gggaaagaac catatctgac caagtcgatg gtgggtccag
     2521 aggatgctgg caactaccgc tgtgtgctag ataccatcaa ccaaggtcat gccaccgtca
     2581 tccgctacga tgtcacagta ttgcccccaa agcattcaga ggaaaaccaa ccaccgaaca
     2641 tcataaccca agaggagcac gagactcctg tccacgtgac tccacagaca ccaccggggc
     2701 aggagccaga gtcggagctg tacccggagc tgcacccaga gttgtacccg gagctcatac
     2761 ctacagtcgc tcaaaatcca gagaaaaaaa tgaaaactcg cctcttaata ctgttgaccc
     2821 ttggctttgt ggttcttgtg gccagcatca ttatctcggt acttcacttt aggaaagtca
     2881 gcgctaaatt gaagaatgca agtgacgaag tcaaacccac cgcgtcagga tccaagtcag
     2941 atcagagttt gtcccaacag atgggattga agaaggcctc tcaggcagat tttaactctg
     3001 actactctgg agataagagc gaggcaacag aaaacacgcg tacgcggccc tcgagtctag
     3061 agggcccgcg gttcgaaggt aagcctatcc ctaaccctct cctcggtctc gattctacgc
     3121 gtaccggtca tcatcaccat caccattgag ccccggggga tccgcccctc tccctccccc
     3181 ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt tgtctatatg
     3241 ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc tggccctgtc
     3301 ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca aggtctgttg
     3361 aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac gtctgtagcg
     3421 accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg ccaaaagcca
     3481 cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt gagttggata
     3541 gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct gaaggatgcc
     3601 cagaaggtac cccattgtat gggatctgat ctggggcctc ggtacacatg ctttacatgt
     3661 gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt ggttttcctt
     3721 tgaaaaacac gatgataata tggccacaac catggttatg ccagagccag cgaagtctgc
     3781 tcccgccccg aaaaagggct ccaagaaggc ggtgactaag gcgcagaaga aaggcggcaa
     3841 gaagcgcaag cgcagccgca aggagagcta ttccatctat gtgtacaagg ttctgaagca
     3901 ggtccaccct gacaccggca tttcgtccaa ggccatgggc atcatgaatt cgtttgtgaa
     3961 cgacattttc gagcgcatcg caggtgaggc ttcccgcctg gcgcattaca acaagcgctc
     4021 gaccatcacc tccagggaga tccagacggc cgtgcgcctg ctgctgcctg gggagttggc
     4081 caagcacgcc gtgtccgagg gtactaaggc catcaccaag tacaccagcg ctaaggatcc
     4141 accggtcgcc accatggcct cctccgagga cgtcatcaag gagttcatgc gcttcaaggt
     4201 gcgcatggag ggctccgtga acggccacga gttcgagatc gagggcgagg gcgagggccg
     4261 cccctacgag ggcacccaga ccgccaagct gaaggtgacc aagggcggcc ccctgccctt
     4321 cgcctgggac atcctgtccc ctcagttcca gtacggctcc aaggcctacg tgaagcaccc
     4381 cgccgacatc cccgactact tgaagctgtc cttccccgag ggcttcaagt gggagcgcgt
     4441 gatgaacttc gaggacggcg gcgtggtgac cgtgacccag gactcctccc tgcaggacgg
     4501 cgagttcatc tacaaggtga agctgcgcgg caccaacttc ccctccgacg gccccgtaat
     4561 gcagaagaag accatgggct gggaggcctc caccgagcgg atgtaccccg aggacggcgc
     4621 cctgaagggc gagatcaaga tgaggctgaa gctgaaggac ggcggccact acgacgccga
     4681 ggtcaagacc acctacatgg ccaagaagcc cgtgcagctg cccggcgcct acaagaccga
     4741 catcaagctg gacatcacct cccacaacga ggactacacc atcgtggaac agtacgagcg
     4801 cgccgagggc cgccactcca ccggcgccta agcggccgca attcactcct caggtgcagg
     4861 ctgcctatca gaaggtggtg gctggtgtgg ccaatgccct ggctcacaaa taccactgag
     4921 atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag catctgactt
     4981 ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt ttgtgtctct
     5041 cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag tatttggttt
     5101 agagtttggc aacatatgcc catatgctgg ctgccatgaa caaaggttgg ctataaagag
     5161 gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga aaagccttga
     5221 cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta acatccctaa
     5281 aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta ctcccagtca
     5341 tagctgtccc tcttctctta tggagatccc tcgacctgca ccgtcgacca gctggtcgac
     5401 ggtgcaccgt cgaccagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt
     5461 tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt
     5521 gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg
     5581 ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata gtcccgcccc
     5641 taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct
     5701 gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag ctattccaga
     5761 agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact tgtttattgc
     5821 agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt
     5881 ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctggat
     5941 ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct
     6001 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca
     6061 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac
     6121 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt
     6181 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg
     6241 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc
     6301 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
     6361 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc
     6421 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac
     6481 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt
     6541 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct
     6601 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc
     6661 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
     6721 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg
     6781 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc
     6841 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa
     6901 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag
     6961 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg
     7021 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
     7081 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag
     7141 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa
     7201 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc
     7261 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca
     7321 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg
     7381 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
     7441 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc
     7501 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg
     7561 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg
     7621 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt
     7681 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca
     7741 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
     7801 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac
     7861 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa
     7921 gtgccacctg
//
