LOCUS       sequence-481105-        8351 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 8351)
  AUTHORS   Moi D, Nishio S, Li X, Valansi C, Langleib M, Brukman NG, Flyak K, 
            Dessimoz C, de Sanctis D, Tunyasuvunakool K, Jumper J, Grana M, 
            Romero H, Aguilar PS, Jovine L, Podbilewicz B
  TITLE     Discovery of archaeal fusexins homologous to eukaryotic HAP2/GCS1 
            gamete fusion proteins.
  JOURNAL   Nat Commun. 2022 Jul 6;13(1):3880. doi: 10.1038/s41467-022-31564-1.
  PUBMED    35794124
REFERENCE   2  (bases 1 to 8351)
  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: "Nat 
            Commun."; date: "2022-07-6"; volume: "13(1):3880. doi"; pages: " 
            10.1038/s41467-022-31564-1"
COMMENT     Sequence Label: pCI_AFF-1-FLAG_H2B-RFP
FEATURES             Location/Qualifiers
     source          1..8351
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        5..384
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     intron          647..1663
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     1587..1609
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1671..1690
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             3546..3569
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     misc_feature    join(3586..4160,4161..4163,4164..4172)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3753..3770)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3980..3999
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4179..4556
                     /codon_start=1
                     /gene="HIST1H2BJ"
                     /product="human histone H2B"
                     /label=H2B
                     /translation="MPEPAKSAPAPKKGSKKAVTKAQKKGGKKRKRSRKESYSIYVYKV
                     LKQVHPDTGISSKAMGIMNSFVNDIFERIAGEASRLAHYNKRSTITSREIQTAVRLLLP
                     GELAKHAVSEGTKAITKYTSAK"
     regulatory      4569..4578
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             4575..5252
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="MASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAK
                     LKVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGV
                     VTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKM
                     RLKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHS
                     TGA"
     CDS             4575..5252
                     /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(4705..4723)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     4973..4992
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     5167..5190
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(5343..5362)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    5408..5463
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(5462..5481)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(5854..5870)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5854..5870)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5867..5889)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5878..5894
                     /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(5902..5908,5909..5926,5927..5932))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5947..5968
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        6027..6222
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      6073..6208
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    6228..6362
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6265..6284)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6319..6338
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6430..6447)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6601..7189)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6681..6700)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7360..8151,8152..8220))
                     /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     7983..8002
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8221..8325)
                     /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 gggggggcgc gcgccaggcg gggcggggcg gggcgaggcg
      541 gagaggtgcg gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag
      601 gcggcggcgg cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg
      661 ctgccttcgc cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact
      721 gaccgcgtta ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta
      781 gcgcttggtt taatgacggc ttgtttcttt tctgtggctg cgtgaaagcc ttgaggggct
      841 ccgggagggc cctttgtgcg gggggagcgg ctcggggggt gcgtgcgtgt gtgtgtgcgt
      901 ggggagcgcc gcgtgcggct ccgcgctgcc cggcggctgt gagcgctgcg ggcgcggcgc
      961 ggggctttgt gcgctccgca gtgtgcgcga ggggagcgcg gccgggggcg gtgccccgcg
     1021 gtgcgggggg ggctgcgagg ggaacaaagg ctgcgtgcgg ggtgtgtgcg tgggggggtg
     1081 agcagggggt gtgggcgcgt cggtcgggct gcaacccccc ctgcaccccc ctccccgagt
     1141 tgctgagcac ggcccggctt cgggtgcggg gctccgtacg gggcgtggcg cggggctcgc
     1201 cgtgccgggc ggggggtggc ggcaggtggg ggtgccgggc ggggcggggc cgcctcgggc
     1261 cggggagggc tcgggggagg ggcgcggcgg cccccggagc gccggcggct gtcgaggcgc
     1321 ggcgagccgc agccattgcc ttttatggta atcgtgcgag agggcgcagg gacttccttt
     1381 gtcccaaatc tgtgcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg
     1441 cggggcgaag cggtgcggcg ccggcaggaa ggaaatgggc ggggagggcc ttcgtgcgtc
     1501 gccgcgccgc cgtccccttc tccctctcca gcctcggggc tgtccgcggg gggacggctg
     1561 ccttcggggg ggacggggca gggcggggtt cggcttctgg cgtgtgaccg gcggctctag
     1621 agcctctgct aaccatgttc atgccttctt ctttttccta cagctcctgg gcaacgtgct
     1681 ggttattgtg ctgtctcatc attttggcaa agaattgctc gagagttcga agccttaagt
     1741 acgcgtaggc gcgccttaat taacgtttaa acgctagcat ggtactgtgg caatggtcaa
     1801 tagccgttgc catatgtctt gtgatggtta cagaagctcg tctcagaaga catcacagga
     1861 aacgacgatt tgtatctagt aattttgatg aattttattg tggagaaagt gcacatgctc
     1921 agtcacagtt tgaagaggag cgagaatcaa attcctcaaa agtctcatca gtacacagta
     1981 ctcaattcaa ttggggactt gataatacaa tttgtataaa actccagaat gtggttcatg
     2041 ttttaaaata cgaacgcctc gaacaaagat atcctattga aaactcctac acattttcgg
     2101 ttccattaat tgacacaaac tgtaaatgtc attgttatgg ttttgggaca aatgacgttt
     2161 gcaatgtgga aaagtacgct gacgacagaa attgtacgac gagctcagag tttcctacat
     2221 gttacaccaa atatcaccca gcagttgagc cactagattg tccagttaca agtattcctg
     2281 ccaaggcatg ctgcgacatc aagttaaaac cacgcgatgg cagaatgttc cgagctgtga
     2341 aacttcagca accaatcaat gacatgataa tttctcattc tatttttgca aacaacagtg
     2401 gaaaaatgat gaaagttctg ggaccagatg aatttaggat aaatcttctg aaggggaagg
     2461 aacaatttga gttaactgaa taccacagaa tatccgttca attagttgca tcttctcctc
     2521 aacaacaact tcgcgaaggg atgtattatt ttccagagga aaaccacaac gatctgcgtg
     2581 agggtaaaat taatgaaata actgaaagtg atttggataa acttggatgg tatagaagag
     2641 ttggaaatga ttggcaagtt gctacaagtg gattactact tagaaatgca cataaagtgg
     2701 tgataaaaaa ttgcaaagga caagttcata tggatcaatt ttccggaacc aaaaactttg
     2761 ttttacgtgg aactcagtat aacgatactt acaatgaacg aagagtatct gacaataatt
     2821 ttgtaagaag tgtgaaagtt gacgagtctt ctcgagaaat tacaattgtt catgaacatg
     2881 gaaccgctgc acaagtttct ctgaaaactg acactcgtcc aaatctaaca aaaagtcaat
     2941 cacttttggc aaacttcact ggaagtatca cgttagatca tgatggaaat cgaatgctta
     3001 atgtcacttt ctttggcgtc aaaggaactg ttcatatcaa aatgtatgtc aacgatcgaa
     3061 agctcatcgc gacttttgca tgtactgctc aatttggaac gtctctgaaa gatgatggta
     3121 gtagaataag tcttccatcg actataaatc aagctcaatg ggtgtgtatt cttcccgatg
     3181 agcagccaac aaaatcagaa atatgcaaat ggattccata tgaggaaaaa gcaatgagaa
     3241 ctccgagaca agaacaaagt tggtcaaaag gacattcacc gtgctcacaa gcagaatgta
     3301 atagtctgaa aagtggagtg agcgacttgt tcccatggat tatgaatttt gattatttta
     3361 tggctcatgg tggggatttc acagagtggc taaaaattgg aatccatata gtgatagctg
     3421 ttggtctcct gttattatta attcttctct tcacaaaatg ccttgttccg ttggcatgtt
     3481 gctccctgtc aataccattt aaaaatagga acaaaaagaa gaaaaagaag aattcatctg
     3541 attacgacta caaggacgac gatgacaaat aacccggggg atccgcccct ctccctcccc
     3601 cccccctaac gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat
     3661 gttattttcc accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt
     3721 cttcttgacg agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt
     3781 gaatgtcgtg aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc
     3841 gaccctttgc aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc
     3901 acgtgtataa gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat
     3961 agttgtggaa agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc
     4021 ccagaaggta ccccattgta tgggatctga tctggggcct cggtacacat gctttacatg
     4081 tgtttagtcg aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct
     4141 ttgaaaaaca cgatgataat atggccacaa ccatggttat gccagagcca gcgaagtctg
     4201 ctcccgcccc gaaaaagggc tccaagaagg cggtgactaa ggcgcagaag aaaggcggca
     4261 agaagcgcaa gcgcagccgc aaggagagct attccatcta tgtgtacaag gttctgaagc
     4321 aggtccaccc tgacaccggc atttcgtcca aggccatggg catcatgaat tcgtttgtga
     4381 acgacatttt cgagcgcatc gcaggtgagg cttcccgcct ggcgcattac aacaagcgct
     4441 cgaccatcac ctccagggag atccagacgg ccgtgcgcct gctgctgcct ggggagttgg
     4501 ccaagcacgc cgtgtccgag ggtactaagg ccatcaccaa gtacaccagc gctaaggatc
     4561 caccggtcgc caccatggcc tcctccgagg acgtcatcaa ggagttcatg cgcttcaagg
     4621 tgcgcatgga gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc
     4681 gcccctacga gggcacccag accgccaagc tgaaggtgac caagggcggc cccctgccct
     4741 tcgcctggga catcctgtcc cctcagttcc agtacggctc caaggcctac gtgaagcacc
     4801 ccgccgacat ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg
     4861 tgatgaactt cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg
     4921 gcgagttcat ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa
     4981 tgcagaagaa gaccatgggc tgggaggcct ccaccgagcg gatgtacccc gaggacggcg
     5041 ccctgaaggg cgagatcaag atgaggctga agctgaagga cggcggccac tacgacgccg
     5101 aggtcaagac cacctacatg gccaagaagc ccgtgcagct gcccggcgcc tacaagaccg
     5161 acatcaagct ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgagc
     5221 gcgccgaggg ccgccactcc accggcgcct aagcggccgc aattcactcc tcaggtgcag
     5281 gctgcctatc agaaggtggt ggctggtgtg gccaatgccc tggctcacaa ataccactga
     5341 gatctttttc cctctgccaa aaattatggg gacatcatga agccccttga gcatctgact
     5401 tctggctaat aaaggaaatt tattttcatt gcaatagtgt gttggaattt tttgtgtctc
     5461 tcactcggaa ggacatatgg gagggcaaat catttaaaac atcagaatga gtatttggtt
     5521 tagagtttgg caacatatgc ccatatgctg gctgccatga acaaaggttg gctataaaga
     5581 ggtcatcagt atatgaaaca gccccctgct gtccattcct tattccatag aaaagccttg
     5641 acttgaggtt agattttttt tatattttgt tttgtgttat ttttttcttt aacatcccta
     5701 aaattttcct tacatgtttt actagccaga tttttcctcc tctcctgact actcccagtc
     5761 atagctgtcc ctcttctctt atggagatcc ctcgacctgc accgtcgacc agctggtcga
     5821 cggtgcaccg tcgaccagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     5881 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     5941 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     6001 gggaaacctg tcgtgccagc ggatccgcat ctcaattagt cagcaaccat agtcccgccc
     6061 ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc gccccatggc
     6121 tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga gctattccag
     6181 aagtagtgag gaggcttttt tggaggccta ggcttttgca aaaagctaac ttgtttattg
     6241 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     6301 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     6361 tccgctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     6421 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     6481 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     6541 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     6601 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     6661 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     6721 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     6781 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     6841 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     6901 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     6961 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     7021 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     7081 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     7141 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     7201 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     7261 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     7321 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     7381 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     7441 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     7501 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     7561 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     7621 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     7681 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     7741 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     7801 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     7861 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     7921 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     7981 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     8041 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     8101 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     8161 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     8221 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     8281 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     8341 agtgccacct g
//
