LOCUS       sequence-481142-        7580 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 7580)
  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 7580)
  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_GFPnes
FEATURES             Location/Qualifiers
     source          1..7580
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          646..1662
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1586..1608
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1670..1689
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3062..3103
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             3113..3130
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    join(3149..3723,3724..3726,3727..3735)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3316..3333)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3543..3562
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3736..3738,3739..3741,3742..4449)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     CDS             join(3736..3738,3739..3741,3742..4449)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     primer_bind     complement(3781..3802)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4042..4061)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     4389..4410
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4572..4591)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4637..4692
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4691..4710)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5083..5099)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5083..5099)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5096..5118)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5107..5123
                     /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(5131..5137,5138..5155,5156..5161))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5176..5197
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        5256..5451
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      5302..5437
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    5457..5591
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5494..5513)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5548..5567
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5659..5676)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5830..6418)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5910..5929)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6589..7380,7381..7449))
                     /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     7212..7231
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7450..7554)
                     /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 ggggggcgcg cgccgggcgg gggggggcgg ggcgaggcgg
      541 agaggtgcgg cggcagccaa tcagagcggc gcgctccgaa agtttccttt tatggcgagg
      601 cggcggcggc ggcggcccta taaaaagcga agcgcgcggc gggcgggagt cgctgcgcgc
      661 tgccttcgcc ccgtgccccg ctccgccgcc gcctcgcgcc gcccgccccg gctctgactg
      721 accgcgttac tcccacaggt gagcgggcgg gacggccctt ctcctccggg ctgtaattag
      781 cgcttggttt aatgacggct tgtttctttt ctgtggctgc gtgaaagcct tgaggggctc
      841 cgggagggcc ctttgtgcgg ggggagcggc tcggggggtg cgtgcgtgtg tgtgtgcgtg
      901 gggagcgccg cgtgcggctc cgcgctgccc ggcggctgtg agcgctgcgg gcgcggcgcg
      961 gggctttgtg cgctccgcag tgtgcgcgag gggagcgcgg ccgggggcgg tgccccgcgg
     1021 tgcggggggg gctgcgaggg gaacaaaggc tgcgtgcggg gtgtgtgcgt gggggggtga
     1081 gcagggggtg tgggcgcgtc ggtcgggctg caaccccccc tgcacccccc tccccgagtt
     1141 gctgagcacg gcccggcttc gggtgcgggg ctccgtacgg ggcgtggcgc ggggctcgcc
     1201 gtgccgggcg gggggtggcg gcaggtgggg gtgccgggcg gggcggggcc gcctcgggcc
     1261 ggggagggct cgggggaggg gcgcggcggc ccccggagcg ccggcggctg tcgaggcgcg
     1321 gcgagccgca gccattgcct tttatggtaa tcgtgcgaga gggcgcaggg acttcctttg
     1381 tcccaaatct gtgcggagcc gaaatctggg aggcgccgcc gcaccccctc tagcgggcgc
     1441 ggggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg
     1501 ccgcgccgcc gtccccttct ccctctccag cctcggggct gtccgcgggg ggacggctgc
     1561 cttcgggggg gacggggcag ggcggggttc ggcttctggc gtgtgaccgg cggctctaga
     1621 gcctctgcta accatgttca tgccttcttc tttttcctac agctcctggg caacgtgctg
     1681 gttattgtgc tgtctcatca ttttggcaaa gaattgctcg agagttcgaa gccttaagta
     1741 cgcgtaggcg cgccttaatt aacgtttaaa cgctagcgaa ttcggtcgac tggatccggt
     1801 accgaggaga tctgccgccg cgatcgccat ggggccgcat tttacactct tgctggcagc
     1861 tcttgccaac tgcctgtgtc cagggaggcc ctgcatcaaa tgtgaccagt ttgtgacaga
     1921 tgcgctaaag actttcgaaa acacttacct gaatgaccac ctgccacacg acattcacaa
     1981 aaatgtaatg aggatggtga accatgaagt atcgagcttc ggcgtagtca cttcggctga
     2041 ggattcctat ttgggggccg tggacgagaa cacactggaa caagcaacct ggagttttct
     2101 gaaggatctg aagcgtatta cagacagtga cttaaaagga gagctcttta taaaggaact
     2161 attgtggatg cttcgtcatc aaaaggacat ctttaacaat cttgctagac agttccaaaa
     2221 ggaagttctt tgtcccaaca aatgcggagt gatgtcgcag actttgatct ggtgtcttaa
     2281 gtgcgaaaag cagttgcaca tttgtcggaa atccctagat tgtggagagc gccacataga
     2341 ggtacatcgc tcggaagacc tggtcctgga ctgtctgctc agttggcatc gtgcttctaa
     2401 gggacttaca gattacagtt tttacagggt ttgggagaac agttctgaga ccttgattgc
     2461 caaggggaaa gaaccatatc tgaccaagtc gatggtgggt ccagaggatg ctggcaacta
     2521 ccgctgtgtg ctagatacca tcaaccaagg tcatgccacc gtcatccgct acgatgtcac
     2581 agtattgccc ccaaagcatt cagaggaaaa ccaaccaccg aacatcataa cccaagagga
     2641 gcacgagact cctgtccacg tgactccaca gacaccaccg gggcaggagc cagagtcgga
     2701 gctgtacccg gagctgcacc cagagttgta cccggagctc atacctacag tcgctcaaaa
     2761 tccagagaaa aaaatgaaaa ctcgcctctt aatactgttg acccttggct ttgtggttct
     2821 tgtggccagc atcattatct cggtacttca ctttaggaaa gtcagcgcta aattgaagaa
     2881 tgcaagtgac gaagtcaaac ccaccgcgtc aggatccaag tcagatcaga gtttgtccca
     2941 acagatggga ttgaagaagg cctctcaggc agattttaac tctgactact ctggagataa
     3001 gagcgaggca acagaaaaca cgcgtacgcg gccctcgagt ctagagggcc cgcggttcga
     3061 aggtaagcct atccctaacc ctctcctcgg tctcgattct acgcgtaccg gtcatcatca
     3121 ccatcaccat tgagccccgg gggatccgcc cctctccctc ccccccccct aacgttactg
     3181 gccgaagccg cttggaataa ggccggtgtg cgtttgtcta tatgttattt tccaccatat
     3241 tgccgtcttt tggcaatgtg agggcccgga aacctggccc tgtcttcttg acgagcattc
     3301 ctaggggtct ttcccctctc gccaaaggaa tgcaaggtct gttgaatgtc gtgaaggaag
     3361 cagttcctct ggaagcttct tgaagacaaa caacgtctgt agcgaccctt tgcaggcagc
     3421 ggaacccccc acctggcgac aggtgcctct gcggccaaaa gccacgtgta taagatacac
     3481 ctgcaaaggc ggcacaaccc cagtgccacg ttgtgagttg gatagttgtg gaaagagtca
     3541 aatggctctc ctcaagcgta ttcaacaagg ggctgaagga tgcccagaag gtaccccatt
     3601 gtatgggatc tgatctgggg cctcggtaca catgctttac atgtgtttag tcgaggttaa
     3661 aaaaacgtct aggccccccg aaccacgggg acgtggtttt cctttgaaaa acacgatgat
     3721 aatatggcca caaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc
     3781 ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag
     3841 ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc
     3901 gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt cagccgctac
     3961 cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag
     4021 gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga ggtgaagttc
     4081 gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc
     4141 aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta tatcatggcc
     4201 gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat cgaggacggc
     4261 agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     4321 ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc caacgagaag
     4381 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct cggcatggac
     4441 gagctgtaca tgagcggcgg cctgcagaag aagctggagg agctggagct ggactaatgt
     4501 acaagtaaag cggccggccg ccagcacagt ggtcgatcga ccaatgccct ggctcacaaa
     4561 taccactgag atctttttcc ctctgccaaa aattatgggg acatcatgaa gccccttgag
     4621 catctgactt ctggctaata aaggaaattt attttcattg caatagtgtg ttggaatttt
     4681 ttgtgtctct cactcggaag gacatatggg agggcaaatc atttaaaaca tcagaatgag
     4741 tatttggttt agagtttggc aacatatgcc catatgctgg ctgccatgaa caaaggttgg
     4801 ctataaagag gtcatcagta tatgaaacag ccccctgctg tccattcctt attccataga
     4861 aaagccttga cttgaggtta gatttttttt atattttgtt ttgtgttatt tttttcttta
     4921 acatccctaa aattttcctt acatgtttta ctagccagat ttttcctcct ctcctgacta
     4981 ctcccagtca tagctgtccc tcttctctta tggagatccc tcgacctgca ccgtcgacca
     5041 gctggtcgac ggtgcaccgt cgaccagctt ggcgtaatca tggtcatagc tgtttcctgt
     5101 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     5161 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     5221 tttccagtcg ggaaacctgt cgtgccagcg gatccgcatc tcaattagtc agcaaccata
     5281 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     5341 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     5401 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctaact
     5461 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaat ttcacaaata
     5521 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaat gtatcttatc
     5581 atgtctggat ccgctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat
     5641 tgggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
     5701 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc
     5761 aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
     5821 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
     5881 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc
     5941 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
     6001 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
     6061 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt
     6121 atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
     6181 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
     6241 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa
     6301 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
     6361 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
     6421 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg
     6481 gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
     6541 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
     6601 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact
     6661 ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat
     6721 gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg
     6781 aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg
     6841 ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat
     6901 tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc
     6961 ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt
     7021 cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc
     7081 agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
     7141 gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc
     7201 gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa
     7261 acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta
     7321 acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg
     7381 agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg
     7441 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
     7501 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
     7561 tccccgaaaa gtgccacctg
//
