LOCUS       sequence-148198-        5432 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  GCaMP5 under the Toxoplasma SAG1 5' UTR. No Toxoplasma selectable 
            marker.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5432)
  AUTHORS   Sidik SM, Hortua Triana MA, Paul AS, El Bakkouri M, Hackett CG, Tran
            F, Westwood NJ, Hui R, Zuercher WJ, Duraisingh MT, Moreno SN, 
            Lourido S
  TITLE     Using a Genetically Encoded Sensor to Identify Inhibitors of 
            Toxoplasma gondii Ca2+ Signaling.
  JOURNAL   J Biol Chem. 2016 Apr 29;291(18):9566-80. doi: 
            10.1074/jbc.M115.703546. Epub 2016 Mar 1.
  PUBMED    26933036
REFERENCE   2  (bases 1 to 5432)
  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: 
            "10.1074/jbc.M115.703546"; journalName: "J Biol Chem"; date: 
            "2016-04-29- 29"; volume: "291"; issue: "18"; pages: "9566-80"
COMMENT     Sequence Label: pGCaMP5
FEATURES             Location/Qualifiers
     source          1..5432
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     669..685
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     670..686
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(1517..1525,1526..1543,1544..1546,1547..1579,1580..1582,
                     1583..1606,1607..1633,1634..1690,1691..1696,1697..1966,
                     1967..1984,1985..2419,2420..2425,2426..2869)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     CDS             join(1517..1525,1526..1543,1544..1546,1547..1579,1580..1582,
                     1583..1606,1607..1633,1634..1690,1691..1696,1697..1966,
                     1967..1984,1985..2419,2420..2425,2426..2869)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      1613..1622
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1903..1924
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2030..2051)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2291..2310)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     complement(3191..3207)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3191..3207)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(3244..3264)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3244..3262)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3283..3299)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3283..3299)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3296..3318)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3307..3323
                     /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(3331..3337,3338..3355,3356..3361))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3376..3397
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3514..3531)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3685..4273)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3765..3784)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4444..5235,5236..5304))
                     /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     5067..5086
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5305..5409)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgacg gtatcgataa gcttttacat ccgttgcctt ttccacggtc
      721 cgtgatttca tgtgcgtgca gcttcaaaga ctggtcgttg cgactaataa gactgcagtg
      781 acaggtcgaa tggtgggcac cttgctgatg actatctact gcaaagtctg agacaacgaa
      841 cgaaacttcc cacacgaggc atttgaaact gacggtgtct aggtaatatg cactgcaaga
      901 cacggtactg gggcctcgct gaattagggg ccgatctcgt tgccctatca gtgctcacag
      961 tgccgcaacg taacaccagg gcaggttctt gacagtggca acaatgtgcg acgggcgtgt
     1021 gaacgtttcg tagtcatagc gctagcacgt acctagccac atggtcgtga ggagctttac
     1081 catgcgtcta gaaggtggat gcgggacacg ccttcctggc ctttggctcc cgagacgcgt
     1141 gttctaacca caaaccttga gacgcgtgtt ccaaccacgc accctgacac gcgtgttcca
     1201 accacgcacc ctgagacgcg tgttctaacc acgcaccctg agacgcgtgt tctaaccacg
     1261 caccctgaga cgcgtgttct gccgcacaat gtgcacctgt aggaagctgt agtcactgct
     1321 gattctcact gttctcggca agggccgacg accggagtac agtttttgtg ggcagagccg
     1381 ttgtgcagct ttccgttctt ctcggttgtg tcacatgtgt cattgtcgtg taaacacacg
     1441 gttgtatgtc ggtttcgctg caccacttca ttatttcttc tggttttttg acgagtatgc
     1501 atcctttttc gacaaaatgg gttctcatca tcatcatcat catggtatgg ctagcatgac
     1561 tggtggacag caaatgggtc gggatctgta cgacgatgac gataaggatc tcgccaccat
     1621 ggtcgactca tcacgtcgta agtggaataa gacaggtcac gcagtcagag ctataggtcg
     1681 gctgagctca ctcgagaacg tctatatcaa ggccgacaag cagaagaacg gcatcaaggc
     1741 gaacttcaag atccgccaca acatcgagga cggcggcgtg cagctcgcct accactacca
     1801 gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcgt
     1861 gcagtccaaa ctttcgaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt
     1921 cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagggcg gtaccggagg
     1981 gagcatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc tggtcgagct
     2041 ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggtgagg gcgatgccac
     2101 ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg tgccctggcc
     2161 caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc ccgaccacat
     2221 gaagcagcac gacttcttca agtccgccat gcccgaaggc tacatccagg agcgcaccat
     2281 cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg agggcgacac
     2341 cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca acatcctggg
     2401 gcacaagctg gagtacaacc tgccggacca actgactgaa gagcagatcg cagaatttaa
     2461 agaggctttc tccctatttg acaaggacgg ggatgggaca ataacaacca aggagctggg
     2521 gacggtgatg cggtctctgg ggcagaaccc cacagaagca gagctgcagg acatgatcaa
     2581 tgaagtagat gccgacggtg acggcacaat cgacttccct gagttcctga caatgatggc
     2641 aagaaaaatg aaatacacag acagtgaaga agaaattaga gaagcgttcc gtgtgtttga
     2701 taaggatggc aatggctaca tcagtgcagc agagcttcgc cacgtgatga caaaccttgg
     2761 agagaagtta acagatgaag aggttgatga aatgatcagg gaagcagaca tcgatgggga
     2821 tggtcaggta aactacgaag agtttgtaca aatgatgaca gcgaagtgat taattaatca
     2881 ccgttgtgct cacttctcaa atcgacaaag gaaacacact tcgtgcagca tgtgccccat
     2941 tataaagaaa ctgagttgtt ccgttgtggc ttgcaggtgt cacatccaca aaaaccggcc
     3001 gactctaaat aggagtgttt cgcagcaagc agcgaaagtt tatgactggg tccgaatctc
     3061 tgaacggatg tgtggcggac ctggctgatg ttgatcgccg tcgacacacg cgccacatgg
     3121 gtcaatacac aagacagcta tcagttgttt tagtcgaacc ggttaacaca attcttgccc
     3181 ccccgagggg gatccactag ttctagagcg gccgccaccg cggtggagct ccagcttttg
     3241 ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt
     3301 gtgaaattgt tatccgctca caattccaca caacatacga gccggaagca taaagtgtaa
     3361 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     3421 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3481 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3541 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3601 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     3661 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     3721 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     3781 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     3841 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     3901 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     3961 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4021 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4081 tacagagttc ttgaagtggt ggcctaacta cggctacact agaaggacag tatttggtat
     4141 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4201 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4261 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4321 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4381 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4441 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4501 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4561 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     4621 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     4681 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     4741 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     4801 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     4861 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     4921 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     4981 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5041 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5101 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5161 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5221 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5281 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5341 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5401 ggttccgcgc acatttcccc gaaaagtgcc ac
//
