LOCUS       sequence-324260-        5607 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Allows for transcription of mCerulean fluorophore half fused to 
            TDP-43 for injection into zebrafish embryos for BiFC assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5607)
  AUTHORS   Don EK, Maschirow A, Radford RAW, Scherer NM, Vidal-Itriago A, Hogan
            A, Maurel C, Formella I, Stoddart JJ, Hall TE, Lee A, Shi B, Cole 
            NJ, Laird AS, Badrock AP, Chung RS, Morsch M
  TITLE     In vivo Validation of Bimolecular Fluorescence Complementation 
            (BiFC) to Investigate Aggregate Formation in Amyotrophic Lateral 
            Sclerosis (ALS).
  JOURNAL   Mol Neurobiol. 2021 May;58(5):2061-2074. doi: 
            10.1007/s12035-020-02238-0. Epub 2021 Jan 7.
  PUBMED    33415684
REFERENCE   2  (bases 1 to 5607)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1007/s12035-020-02238-0"; journalName: "Mol Neurobiol"; date: 
            "2021-05"; volume: "58"; issue: "5"; pages: "2061-2074"
COMMENT     Sequence Label: pCS2plus-mCerulean 156-239-GGGS-TDP-43
FEATURES             Location/Qualifiers
     source          1..5607
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     regulatory      88..97
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     polyA_signal    1659..1793
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1683..1702)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1737..1756
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(1906..1926)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1906..1924)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1945..1961)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1945..1961)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1958..1980)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1969..1985
                     /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(1993..1999,2000..2017,2018..2023))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2038..2059
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2176..2193)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2347..2935)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2427..2446)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3106..3897,3898..3966))
                     /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     3729..3748
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3967..4071)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(4097..4552)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4234..4255)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     4446..4465
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4623..5607
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atccgccgcc accatggaca agcagaagaa cggaatcaag
      121 gccaacttca agatccgcca caacatcgag gacggaagcg tgcagctggc cgaccactac
      181 cagcagaaca cccctatcgg agatggacct gtgctgctgc cagacaacca ctacctgagc
      241 acccagagca agctgagcaa ggaccctaac gagaagcgcg accacatggt gctgctggaa
      301 tttgtgaccg ccgctggaat caccctggga atggacgagc tgtacaaggg cggaggaagc
      361 ggaggcggaa gcggcggggg aagcatgtct gaatatattc gggtaaccga agatgagaac
      421 gatgagccca ttgaaatacc atcggaagac gatgggacgg tgctgctctc cacggttaca
      481 gcccagtttc caggggcgtg tgggcttcgc tacaggaatc cagtgtctca gtgtatgaga
      541 ggtgtccggc tggtagaagg aattctgcat gccccagatg ctggctgggg aaatctggtg
      601 tatgttgtca actatccaaa agataacaaa agaaaaatgg atgagacaga tgcttcatca
      661 gcagtgaaag tgaaaagagc agtccagaaa acatccgatt taatagtgtt gggtctccca
      721 tggaaaacaa ccgaacagga cctgaaagag tattttagta cctttggaga agttcttatg
      781 gtgcaggtca agaaagatct taagactggt cattcaaagg ggtttggctt tgttcgtttt
      841 acggaatatg aaacacaagt gaaagtaatg tcacagcgac atatgataga tggacgatgg
      901 tgtgactgca aacttcctaa ttctaagcaa agccaagatg agcctttgag aagcagaaaa
      961 gtgtttgtgg ggcgctgtac agaggacatg actgaggatg agctgcggga gttcttctct
     1021 cagtacgggg atgtgatgga tgtcttcatc cccaagccat tcagggcctt tgcctttgtt
     1081 acatttgcag atgatcagat tgcgcagtct ctttgtggag aggacttgat cattaaagga
     1141 atcagcgttc atatatccaa tgccgaacct aagcacaata gcaatagaca gttagaaaga
     1201 agtggaagat ttggtggtaa tccaggtggc tttgggaatc agggtggatt tggtaatagc
     1261 agagggggtg gagctggttt gggaaacaat caaggtagta atatgggtgg tgggatgaac
     1321 tttggtgcgt tcagcattaa tccagccatg atggctgccg cccaggcagc actacagagc
     1381 agttggggta tgatgggcat gttagccagc cagcagaacc agtcaggccc atcgggtaat
     1441 aaccaaaacc aaggcaacat gcagagggag ccaaaccagg ccttcggttc tggaaataac
     1501 tcttatagtg gctctaattc tggtgcagca attggttggg gatcagcatc caatgcaggg
     1561 tcgggcagtg gttttaatgg aggctttggc tcaagcatgg attctaagtc ttctggctgg
     1621 ggaatgtagt ctagaactat agtgagtcgt attacgtaga tccagacatg ataagataca
     1681 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     1741 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     1801 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaattcg
     1861 cggccgcggc gccaatgcat tgggcccggt acccagcttt tgttcccttt agtgagggtt
     1921 aattgcgcgc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt gttatccgct
     1981 cacaattcca cacaacatac gagccgggag cataaagtgt aaagcctggg gtgcctaatg
     2041 agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct
     2101 gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
     2161 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
     2221 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
     2281 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
     2341 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
     2401 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
     2461 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
     2521 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
     2581 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
     2641 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
     2701 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
     2761 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
     2821 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
     2881 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     2941 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     3001 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     3061 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     3121 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     3181 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     3241 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     3301 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     3361 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     3421 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     3481 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     3541 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     3601 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     3661 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     3721 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     3781 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     3841 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     3901 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     3961 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     4021 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     4081 ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     4141 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     4201 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     4261 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     4321 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     4381 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     4441 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     4501 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg
     4561 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     4621 cagtcgaccg ccaattcaat atggcgtata tggactcatg ccaattcaat atggtggatc
     4681 tggacctgtg ccaattcaat atggcgtata tggactcgtg ccaattcaat atggtggatc
     4741 tggaccccag ccaattcaat atggcggact tggcaccatg ccaattcaat atggcggacc
     4801 tggcactgtg ccaactgggg aggggtctac ttggcacggt gccaagtttg aggaggggtc
     4861 ttggccctgt gccaagtccg ccatattgaa ttggcatggt gccaataatg gcggccatat
     4921 tggctatatg ccaggatcaa tatataggca atatccaata tggccctatg ccaatatggc
     4981 tattggccag gttcaatact atgtattggc cctatgccat atagtattcc atatatgggt
     5041 tttcctattg acgtagatag cccctcccaa tgggcggtcc catataccat atatggggct
     5101 tcctaatacc gcccatagcc actcccccat tgacgtcaat ggtctctata tatggtcttt
     5161 cctattgacg tcatatgggc ggtcctattg acgtatatgg cgcctccccc attgacgtca
     5221 attacggtaa atggcccgcc tggctcaatg cccattgacg tcaataggac cacccaccat
     5281 tgacgtcaat gggatggctc attgcccatt catatccgtt ctcacgcccc ctattgacgt
     5341 caatgacggt aaatggccca cttggcagta catcaatatc tattaatagt aacttggcaa
     5401 gtacattact attggaagta cgccagggta cattggcagt actcccattg acgtcaatgg
     5461 cggtaaatgg cccgcgatgg ctgccaagta catccccatt gacgtcaatg gggaggggca
     5521 atgacgcaaa tgggcgttcc attgacgtaa atgggcggta ggcgtgccta atgggaggtc
     5581 tatataagca atgctcgttt agggaac
//
