LOCUS       Exported                8480 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  GCaMP5 under the Toxoplasma SAG1 5' UTR. Encodes a DHFR selectable 
            marker..
ACCESSION   .
VERSION     .
KEYWORDS    pGCaMP5-DHFR
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8480)
  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 8480)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8480
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(3..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             1526..1543
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1547..1579
                     /codon_start=1
                     /product="leader peptide from bacteriophage T7 gene 10"
                     /label=T7 tag (gene 10 leader)
                     /note="promotes efficient translation in E. coli"
                     /translation="MASMTGGQQMG"
     CDS             1583..1606
                     /codon_start=1
                     /product="Xpress(TM) epitope tag, including an enterokinase
                     recognition and cleavage site"
                     /label=Xpress(TM) tag
                     /translation="DLYDDDDK"
     regulatory      1613..1622
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1634..1690
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     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"
     CDS             2426..2869
                     /codon_start=1
                     /product="calcium-binding protein"
                     /label=calmodulin
                     /translation="DQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEA
                     ELQDMINEVDADGDGTIDFPEFLTMMARKMKYTDSEEEIREAFRVFDKDGNGYISAAEL
                     RHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     primer_bind     complement(6239..6255)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6239..6255)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     primer_bind     complement(6292..6312)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(6292..6310)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(6331..6347)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6331..6347)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6344..6366)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6355..6371
                     /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(6379..6409)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6424..6445
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6562..6579)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6733..7321)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6813..6832)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7492..8352)
                     /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     8115..8134
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8353..8457)
                     /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 ccgctgtggc 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 gatcgatcca ctagcatgtc attcgatttt caccccccgc gtagttcctg
     3241 tgtgtcattc gttgtcgaga caactctgtc ccgccccggt gctgttccat atgcgtgact
     3301 ttcccgcaat tttttcagac tttcaggaaa gacaggctcc ggaacgatct cgtccatgac
     3361 tggtaaatcc acgacaccgc aatggccccc agcacctcta tctctcgtgc caggggacta
     3421 acgttgtatg cgtctgcgtc ttgtcttttt gcattcgctt tccaaaaaag agagccatcc
     3481 gttcccccgc acattcaacg ccgcgagtgc ggtttttgtc ttttttgagt ggtaggacgc
     3541 ttttcatgcg cgaactacgt ggacattaag ttccattctc tttttcgaca gcacgaaacc
     3601 ttgcattcaa acccgcccgc ggaagatccg atcttgctgc tgttcgcagt cccagtagcg
     3661 tcctgtcggc cgcgccgtct ctgttggtgg gcagccgcta cacctgttat ctgactgccg
     3721 tgcgcgaaaa tgacgccatt tttgggaaaa tcggggaact tcattcttta aaagtatgcg
     3781 gaggtttcct ttttcttctg ttcgtttctt tttctcgggt ttgataaccg tgttcgatgt
     3841 aagcactttc cgtctctcct ccgtgctttg ttcgacatcg agaccaggtg tgcagatcct
     3901 tcgcttgtcg atccggagac gcgtgtctcg tagaaccttt tcattttacc acacggcagt
     3961 gcggagcact gctctgagtg cagcagggac gggtgaagtt tcgctttagt agtgcgtttc
     4021 tgctctacgg ggcgttgtcg tgtctgggaa gatgcagaaa ccggtgtgtc tggtcgtcgc
     4081 gatgaccccc aagaggggca tcggcatcaa caacggcctc ccgtggcccc acttgaccac
     4141 agatttcaaa cactttcgtc gtgtgacaaa aacgacgccc gaagaagcca gtcgcctgaa
     4201 cgggtggctt cccaggaaat ttgcaaagac gggcgactct ggacttccct ctccatcagt
     4261 cggcaagaga ttcaacgccg ttgtcatggg acggaaaaac tgggaaagca tgcctcgaaa
     4321 gtttagaccc ctcgtggaca gattgaacat cgtcgtttcc tcttccctca aagaagaaga
     4381 cattgcggcg gagaagcctc aagctgaagg ccagcagcgc gtccgagtct gtgcttcact
     4441 cccagcagct ctcagccttc tggaggaaga gtacaaggat tctgtcgacc agatttttgt
     4501 cgtgggagga gcgggactgt acgaggcagc gctgtctctg ggcgttgcct ctcacctgta
     4561 catcacgcgt gtagcccgcg agtttccgtg cgacgttttc ttccctgcgt tccccggaga
     4621 tgacattctt tcaaacaaat caactgctgc gcaggctgca gctcctgccg agtctgtgtt
     4681 cgttcccttt tgtccggagc tcggaagaga gaaggacaat gaagcgacgt atcgacccat
     4741 cttcatttcc aagaccttct cagacaacgg ggtaccctac gactccgtgg ttctcgagaa
     4801 gagaaggaag actgacgacg cagccactgc ggaaccgagc aacgcaatga gctccttgac
     4861 gtccacgagg gagacaactc ccgtgcacgg gttgcaggct ccttcttcgg ccgcagccat
     4921 tgccccggtg ttggcgtgga tggacgaaga agaccggaaa aaacgcgagc aaaaggaact
     4981 gattcgggcc gttccgcatg ttcactttag aggccatgaa gaattccagt accttgatct
     5041 cattgccgac attattaaca atggaaggac aatggatgac cgaacgggcg ttggtgtcat
     5101 ctccaaattc ggctgcacta tgcgctactc gctggatcag gcctttccac ttctcaccac
     5161 aaagcgtgtg ttctggaaag gggtcctcga agagttgctg tggttcattc gcggcgacac
     5221 gaacgcaaac catctttctg agaagggcgt gaagatctgg gacaagaatg tgacacgcga
     5281 gttcctcgat tcgcgcaatc tcccccaccg agaggtcgga gacatcggcc cgggctacgg
     5341 cttccagtgg agacacttcg gcgcggcata caaagacatg cacacagact acacagggca
     5401 gggcgtcgac cagctgaaga atgtgatcca gatgctgaga acgaatccaa cagatcgtcg
     5461 catgctcatg actgcctgga atcctgcagc gctggacgaa atggcgctgc cgccttgtca
     5521 cttgttgtgc cagttctacg tgaacgacca gaaggagctg tcgtgcatca tgtatcagcg
     5581 gtcgtgcgat gtcggcctcg gcgtcccctt caacatcgct tcctattcgc ttttgacgct
     5641 catggttgca cacgtctgca acctaaaacc taaggagttc attcacttca tggggaacac
     5701 gcatgtctac acgaaccatg tcgaggcttt aaaagagcag ctgcggagag aaccgagacc
     5761 gttccccatt gtgaacatcc tcaacaagga acgcatcaag gaaatcgacg atttcaccgc
     5821 cgaggatttt gaggtcgtgg gctacgtccc gcacggacga atccagatgg agatggctgt
     5881 ctagcggaaa tacagaagct gcccgtctct cgttttcctc tcttttcgga gggatcaggg
     5941 agagtgcctc gggtcggaga gagctgacga gggggtgcca gagacccctg tgtcctttat
     6001 cgaagaaaag ggatgactct tcatgtggca tttcacacag tctcacctcg ccttgttttc
     6061 tttttgtcaa tcagaacgaa agcgagttgc gggtgacgca gatgtgcgtg tatccactcg
     6121 tgaatgcgtt atcgttctgt atgccgctag agtgctggac tgttgctgtc tgcccacgac
     6181 agcagacaac tttccttcta tgcacttgca ggatggaatt cctgcagccc gggggatcga
     6241 tccactagtt ctagagcggc cgccaccgcg gtggagctcc agcttttgtt ccctttagtg
     6301 agggttaatt gcgcgcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta
     6361 tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc
     6421 ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg
     6481 aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg
     6541 tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     6601 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     6661 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     6721 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     6781 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     6841 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     6901 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     6961 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     7021 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     7081 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     7141 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     7201 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     7261 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     7321 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     7381 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     7441 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     7501 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     7561 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     7621 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     7681 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     7741 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     7801 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     7861 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     7921 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     7981 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     8041 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     8101 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     8161 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     8221 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     8281 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     8341 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     8401 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     8461 atttccccga aaagtgccac
//
