LOCUS       sequence-315190-        4578 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Allows for transcription of improved control mVenus I152L 
            fluorophore half for injection into zebrafish embryos for BiFC 
            assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4578)
  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 4578)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 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-mVenus1-155-I152L-GGGS
FEATURES             Location/Qualifiers
     source          1..4578
                     /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"
     CDS             join(94..96,97..99,100..558)
                     /codon_start=1
                     /product="improved N-terminal fragment of mVenus for use in
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN155(I152L)
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYLTA"
     primer_bind     complement(139..160)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(400..419)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    627..761
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(651..670)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     705..724
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(874..894)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(874..892)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(913..929)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(913..929)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(926..948)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    937..953
                     /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(961..967,968..985,986..991))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1006..1027
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1144..1161)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1315..1903)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1395..1414)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2074..2865,2866..2934))
                     /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     2697..2716
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2935..3039)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3065..3520)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3202..3223)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3414..3433
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3594..4578
                     /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 accatggtga gcaagggcga ggagctgttc
      121 accggggtgg tgcccatcct ggtcgagctg gacggcgacg taaacggcca caagttcagc
      181 gtgtccggcg agggcgaggg cgatgccacc tacggcaagc tgaccctgaa gctgatctgc
      241 accaccggca agctgcccgt gccctggccc accctcgtga ccaccctggg ctacggcctg
      301 cagtgcttcg cccgctaccc cgaccacatg aagcagcacg acttcttcaa gtccgccatg
      361 cccgaaggct acgtccagga gcgcaccatc ttcttcaagg acgacggcaa ctacaagacc
      421 cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc gcatcgagct gaagggcatc
      481 gacttcaagg aggacggcaa catcctgggg cacaagctgg agtacaacta caacagccac
      541 aacgtctatc tcaccgccgg cggaggaagc ggaggcggaa gcggcggggg aagctaatct
      601 agaactatag tgagtcgtat tacgtagatc cagacatgat aagatacatt gatgagtttg
      661 gacaaaccac aactagaatg cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta
      721 ttgctttatt tgtaaccatt ataagctgca ataaacaagt taacaacaac aattgcattc
      781 attttatgtt tcaggttcag ggggaggtgt gggaggtttt ttaattcgcg gccgcggcgc
      841 caatgcattg ggcccggtac ccagcttttg ttccctttag tgagggttaa ttgcgcgctt
      901 ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca caattccaca
      961 caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag tgagctaact
     1021 cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct
     1081 gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc
     1141 ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca
     1201 ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg
     1261 agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca
     1321 taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa
     1381 cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc
     1441 tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc
     1501 gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct
     1561 gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg
     1621 tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag
     1681 gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta
     1741 cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg
     1801 aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt
     1861 tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt
     1921 ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag
     1981 attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat
     2041 ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc
     2101 tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat
     2161 aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc
     2221 acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag
     2281 aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag
     2341 agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt
     2401 ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg
     2461 agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt
     2521 tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc
     2581 tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc
     2641 attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa
     2701 taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg
     2761 aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc
     2821 caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag
     2881 gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt
     2941 cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt
     3001 tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc
     3061 acctaaattg taagcgttaa tattttgtta aaattcgcgt taaatttttg ttaaatcagc
     3121 tcatttttta accaataggc cgaaatcggc aaaatccctt ataaatcaaa agaatagacc
     3181 gagatagggt tgagtgttgt tccagtttgg aacaagagtc cactattaaa gaacgtggac
     3241 tccaacgtca aagggcgaaa aaccgtctat cagggcgatg gcccactacg tgaaccatca
     3301 ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac taaatcggaa ccctaaaggg
     3361 agcccccgat ttagagcttg acggggaaag ccggcgaacg tggcgagaaa ggaagggaag
     3421 aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag cggtcacgct gcgcgtaacc
     3481 accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt cccattcgcc attcaggctg
     3541 cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc tattacgcca gtcgaccata
     3601 gccaattcaa tatggcgtat atggactcat gccaattcaa tatggtggat ctggacctgt
     3661 gccaattcaa tatggcgtat atggactcgt gccaattcaa tatggtggat ctggacccca
     3721 gccaattcaa tatggcggac ttggcaccat gccaattcaa tatggcggac ttggcactgt
     3781 gccaactggg gaggggtcta cttggcacgg tgccaagttt gaggaggggt cttggccctg
     3841 tgccaagtcc gccatattga attggcatgg tgccaataat ggcggccata ttggctatat
     3901 gccaggatca atatataggc aatatccaat atggccctat gccaatatgg ctattggcca
     3961 ggttcaatac tatgtattgg ccctatgcca tatagtattc catatatggg ttttcctatt
     4021 gacgtagata gcccctccca atgggcggtc ccatatacca tatatggggc ttcctaatac
     4081 cgcccatagc cactccccca ttgacgtcaa tggtctctat atatggtctt tcctattgac
     4141 gtcatatggg cggtcctatt gacgtatatg gcgcctcccc cattgacgtc aattacggta
     4201 aatggcccgc ctggctcaat gcccattgac gtcaatagga ccacccacca ttgacgtcaa
     4261 tgggatggct cattgcccat tcatatccgt tctcacgccc cctattgacg tcaatgacgg
     4321 taaatggccc acttggcagt acatcaatat ctattaatag taacttggca agtacattac
     4381 tattggaagg acgccagggt acattggcag tactcccatt gacgtcaatg gcggtaaatg
     4441 gcccgcgatg gctgccaagt acatccccat tgacgtcaat ggggaggggc aatgacgcaa
     4501 atgggcgttc cattgacgta aatgggcggt aggcgtgcct aatgggaggt ctatataagc
     4561 aatgctcgtt tagggaac
//
