LOCUS       sequence-481120-        7088 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 7088)
  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 7088)
  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_GFPnes
FEATURES             Location/Qualifiers
     source          1..7088
                     /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"
     misc_feature    join(2657..3231,3232..3234,3235..3243)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2824..2841)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3051..3070
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             join(3244..3246,3247..3249,3250..3957)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELY"
     CDS             join(3244..3246,3247..3249,3250..3957)
                     /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(3289..3310)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3550..3569)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3897..3918
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(4080..4099)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4145..4200
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4199..4218)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4591..4607)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4591..4607)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4604..4626)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4615..4631
                     /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(4639..4645,4646..4663,4664..4669))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4684..4705
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4764..4959
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4810..4945
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4965..5099
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5002..5021)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5056..5075
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5167..5184)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5338..5926)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5418..5437)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6097..6888,6889..6957))
                     /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     6720..6739
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6958..7062)
                     /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 tatttatttt ttttttaatt atttggggcg
      481 gggggggggg gggggggggg ggggggcgcg cgccaggcgg ggcggggcgg 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 cgctagcctc tttggcatca ggaggagcaa
     1801 caggagggcc ctgccttgaa ggtcatggca cagtggtggc agatcctctt ggggttgtgg
     1861 gcagtcctac ccaccttggc aggggacaaa ctgctcagcg tctgcatgaa ttccaagcgc
     1921 cacaagcaag aacctggccc agaagacgaa ctctaccagg agtgcaggcc ttgggaggac
     1981 aatgcctgct gcacacgttc cacaagttgg gaagcccacc ttgaggagcc cttgctcttt
     2041 aacttcagca tgatgcactg tggactgctg accccggcct gtcgcaagca cttcatccag
     2101 gccatctgct tccatgagtg ttcccccaac ctggggcctt ggatccagcc ggtggtccca
     2161 aacgggcagg aagagcagcg ggtttggggc gtgccgctgt gccaggagga ttgtgaggac
     2221 tggtggcgag cctgccactc atctttgact tgtaaatcca actggctcca tggctgggac
     2281 tggagtgagg agaagaaaca ctgccccgca catgagccct gcctcccttt ttcgtatcac
     2341 ttccccaccc cggatgacct gtgtgagaag atctggaaca acacattcaa ggccagtccc
     2401 gagcgcagga acagtgggcg gtgtctgcag aagtggtttg agcccaccct tagcaacccc
     2461 aacgtggaag tggccctcca ctttgccggc tctgccttag ccccacagct ctcttacacc
     2521 ctcccagcct tctctctgtg cctgttgttc catccctgat ccctgcttcc ctctccaacc
     2581 cacatttcct gatccttctg ggccatggcc atctccatcc tcaggctatt catgttgggg
     2641 gcacccgggg gatccgcccc tctccctccc ccccccctaa cgttactggc cgaagccgct
     2701 tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg ccgtcttttg
     2761 gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct aggggtcttt
     2821 cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca gttcctctgg
     2881 aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg aaccccccac
     2941 ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct gcaaaggcgg
     3001 cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa tggctctcct
     3061 caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt atgggatctg
     3121 atctggggcc tcggtacaca tgctttacat gtgtttagtc gaggttaaaa aaacgtctag
     3181 gccccccgaa ccacggggac gtggttttcc tttgaaaaac acgatgataa tatggccaca
     3241 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     3301 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     3361 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     3421 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     3481 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     3541 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     3601 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     3661 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     3721 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     3781 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     3841 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     3901 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacatg
     3961 agcggcggcc tgcagaagaa gctggaggag ctggagctgg actaatgtac aagtaaagcg
     4021 gccggccgcc agcacagtgg tcgatcgacc aatgccctgg ctcacaaata ccactgagat
     4081 ctttttccct ctgccaaaaa ttatggggac atcatgaagc cccttgagca tctgacttct
     4141 ggctaataaa ggaaatttat tttcattgca atagtgtgtt ggaatttttt gtgtctctca
     4201 ctcggaagga catatgggag ggcaaatcat ttaaaacatc agaatgagta tttggtttag
     4261 agtttggcaa catatgccca tatgctggct gccatgaaca aaggttggct ataaagaggt
     4321 catcagtata tgaaacagcc ccctgctgtc cattccttat tccatagaaa agccttgact
     4381 tgaggttaga ttttttttat attttgtttt gtgttatttt tttctttaac atccctaaaa
     4441 ttttccttac atgttttact agccagattt ttcctcctct cctgactact cccagtcata
     4501 gctgtccctc ttctcttatg gagatccctc gacctgcacc gtcgaccagc tggtcgacgg
     4561 tgcaccgtcg accagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     4621 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     4681 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     4741 aaacctgtcg tgccagcgga tccgcatctc aattagtcag caaccatagt cccgccccta
     4801 actccgccca tcccgcccct aactccgccc agttccgccc attctccgcc ccatggctga
     4861 ctaatttttt ttatttatgc agaggccgag gccgcctcgg cctctgagct attccagaag
     4921 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctaacttg tttattgcag
     4981 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5041 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctggatcc
     5101 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5161 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5221 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5281 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     5341 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     5401 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     5461 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     5521 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     5581 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     5641 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     5701 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     5761 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     5821 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     5881 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     5941 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     6001 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     6061 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     6121 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     6181 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     6241 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     6301 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     6361 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     6421 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     6481 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     6541 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     6601 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     6661 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     6721 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     6781 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     6841 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     6901 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     6961 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     7021 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     7081 gccacctg
//
