LOCUS       Exported                6768 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Fluorescent reporter for cGMP.
ACCESSION   .
VERSION     .
KEYWORDS    pTriEx4-H6-FGB
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6768)
  AUTHORS   Bhargava Y, Hampden-Smith K, Chachlaki K, Wood KC, Vernon J, 
            Allerston CK, Batchelor AM, Garthwaite J
  TITLE     Improved genetically-encoded, FlincG-type fluorescent biosensors for
            neural cGMP imaging.
  JOURNAL   Front Mol Neurosci. 2013 Sep 24;6:26. doi: 10.3389/fnmol.2013.00026.
  PUBMED    24068983
REFERENCE   2  (bases 1 to 6768)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6768
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     misc_recomb     181..1008
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             181..305
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(343..948)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     enhancer        1076..1379
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1381..1580
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1534..1554
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1786..1808
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     1870..1889
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     primer_bind     1931..1950
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1931..1949
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1950..1974
                     /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        1990..2099
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
     CDS             2120..2137
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             2147..2191
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             2207..2224
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             2243..2257
                     /codon_start=1
                     /product="enterokinase recognition and cleavage site"
                     /label=enterokinase site
                     /translation="DDDDK"
     primer_bind     3310..3331
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3437..3458)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3698..3717)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             3911..3943
                     /codon_start=1
                     /product="HSV (herpes simplex virus) epitope tag"
                     /label=HSV tag
                     /translation="QPELAPEDPED"
     CDS             3950..3973
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(4063..4082)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    4128..4183
                     /label=-beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4182..4201)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     terminator      4271..4318
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     misc_recomb     4331..5036
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     3'UTR           4419..4791
                     /label=polyhedrin 3' UTR
                     /note="3' UTR from the Autographa californica 
                     nucleopolyhedrovirus polyhedrin gene (Suzuki et al., 2006)"
     CDS             complement(4448..5036)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="IPKSSTTNLIADVLADTINRRRVAMAKSSSEATSNDEGWDDDDNR
                     PNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVEPLLQNKTQTNIDKAR
                     LLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDLIFKFRYKDAENHLIF
                     ALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     protein_bind    5045..5078
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     rep_origin      complement(5246..5833)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5325..5344)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5846..6706)
                     /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     6469..6488
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 tcctgcatct tttaatcaaa tcccaagatg tgtataaacg cgccggtatg tacaggaaga
       61 ggtttatact aaactgttac attgcaaacg tggtttcgtg tgccaagtgt gaaaaccgat
      121 gtttaatcaa ggctctgacg catttctaca accacgactc caagtgtgtg ggtgaagtca
      181 tgcatctttt aatcaaatcc caagatgtgt ataaaccacc aaactgccaa aaaatgaaaa
      241 ctgtcgacaa gctctgtccg tttgctggca actgcaaggg tctcaatcct atttgtaatt
      301 attgaataat aaaacaatta taaatgtcaa atttgttttt tattaacgat acaaaccaaa
      361 cgcaacaaga acatttgtag tattatctat aattgaaaac gcgtagttat aatcgctgag
      421 gtaatattta aaatcatttt caaatgattc acagttaatt tgcgacaata taattttatt
      481 ttcacataaa ctagacgcct tgtcgtcttc ttcttcgtat tccttctctt tttcattttt
      541 ctcttcataa aaattaacat agttattatc gtatccatat atgtatctat cgtatagagt
      601 aaattttttg ttgtcataaa tatatatgtc ttttttaatg gggtgtatag taccgctgcg
      661 catagttttt ctgtaattta caacagtgct attttctggt agttcttcgg agtgtgttgc
      721 tttaattatt aaatttatat aatcaatgaa tttgggatcg tcggttttgt acaatatgtt
      781 gccggcatag tacgcagctt cttctagttc aattacacca ttttttagca gcaccggatt
      841 aacataactt tccaaaatgt tgtacgaacc gttaaacaaa aacagttcac ctcccttttc
      901 tatactattg tctgcgagca gttgtttgtt gttaaaaata acagccattg taatgagacg
      961 cacaaactaa tatcacaaac tggaaatgtc tatcaatata tagttgctga tggccggcct
     1021 attaatagta atcaattacg gggtcattag ttcatagccc atatatggag ttccgcgtta
     1081 cataacttac ggtaaatggc ccgcctggct gaccgcccaa cgacccccgc ccattgacgt
     1141 caataatgac gtatgttccc atagtaacgc caatagggac tttccattga cgtcaatggg
     1201 tggagtattt acggtaaact gcccacttgg cagtacatca agtgtatcat atgccaagtc
     1261 cgccccctat tgacgtcaat gacggtaaat ggcccgcctg gcattatgcc cagtacatga
     1321 ccttacggga ctttcctact tggcagtaca tctacgtatt agtcatcgct attaccatgc
     1381 tgatgcggtt ttggcagtac accaatgggc gtggatagcg gtttgactca cggggatttc
     1441 caagtctcca ccccattgac gtcaatggga gtttgttttg gcaccaaaat caacgggact
     1501 ttccaaaatg tcgtaataac cccgccccgt tgacgcaaat gggcggtagg cgtgtacggt
     1561 gggaggtcta tataagcaga cgtcgtttag tgaaccgtca gatcactaga tgctttattg
     1621 cggtagttta tcacagttaa attgctaacg ccagtctcga acttaacgtg cagaagttgg
     1681 tcgtgaggca ctgggcaggt aagtatcggg ccctttgtgc ggggggagcg gctcggggct
     1741 gtccgcgggg ggacggctgc cttcgggggg gacggggcag ggcggggttc ggcttctggc
     1801 gtgtgaccgg cggctctaga gcctctgcta accatgttca tgccttcttc tttttcctac
     1861 agctcctggg caacgtgctg gttattgtgc tgtctcatca ttttggcaaa gaattggatc
     1921 ggaccgaaat taatacgact cactataggg gaattgtgag cggataacaa ttccccggag
     1981 ttaatccggg acctttaatt caacccaaca caatatatta tagttaaata agaattatta
     2041 tcaaatcatt tgtatattaa ttaaaatact atactgtaaa ttacatttta tttacaatca
     2101 aaggagatat accatggcac accatcacca ccatcactct tctggtaaag aaaccgctgc
     2161 tgcgaaattt gaacgccagc acatggactc gccaccgcct tctggtctgg tcccccgggg
     2221 cagcgcaggt tctggtacga ttgatgacga cgacaagatg gtactccgac aggcattccg
     2281 gaagttcacc aaatccgaaa ggtccaagga tctcataaag gaggccatcc ttgacaatga
     2341 ctttatgaag aacttggagc tgtcacagat ccaagagatt gtggattgta tgtacccagt
     2401 ggagtacggc aaagacagct gcatcatcaa agaaggagat gtggggtcac tggtgtatgt
     2461 catggaagat ggtaaggttg aagttacaaa agaaggcgtg aagctgtgca caatgggtcc
     2521 tggtaaagtg tttggagagt tggctatcct ttacaactgt acccggacgg cgaccgtcaa
     2581 aactcttgta aatgtgaaac tctgggccat tgatcgacaa tgttttcaga cgataatgat
     2641 gaggacagga cttatcaagc ataccgagta tatggaattt ttaaaaagcg ttccaacatt
     2701 ccagagcctt cctgaagaga tcctcagtaa gcttgctgac gtccttgaag agacccacta
     2761 tgaaaatggg gaatatatca tcaggcaagg tgcaagaggg gacaccttct ttatcatcag
     2821 taaaggaaag gttaatgtca ctcgtgaaga ctcgcccaat gaagacccag tctttcttag
     2881 aaccttagga aaaggagatt ggtttggaga gaaagccttg cagggggaag atgtgagaac
     2941 agcgaatgta attgcggcag aagctgtaac ctgccttgtg atcgacagag actctttcaa
     3001 acatttgatt ggaggattag atgatgtttc taataaagca tatgaagatg cagaagctaa
     3061 ggcaaaatat gaagctgaag ctgctttctt cgccaacctg aagaacgtct atatcatggc
     3121 cgacaagcag aagaacggca tcaaggcgaa cttcaagatc cgccacaaca tcgaggacgg
     3181 cggcgtgcag ctcgcctacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     3241 gctgcccgac aaccactacc tgagcaccca gtccaaactt tcgaaagacc ccaacgagaa
     3301 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     3361 cgagctgtac aagggcggta ccggagggag catggtgagc aagggcgagg agctgttcac
     3421 cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt
     3481 gtccggcgag ggtgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac
     3541 caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca
     3601 gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc
     3661 cgaaggctac atccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg
     3721 cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga
     3781 cttcaaggag gacggcaaca tcctggggca caagctggag tacaacctgt ctgatttaac
     3841 atcattgaca cccttggagt tggaggtttc ggacgagtag gcggccgcac agctgtatac
     3901 acgtgcaagc cagccagaac tcgccccgga agaccccgag gatctcgagc accaccatca
     3961 ccatcaccat cactaagtga ttaacctcag gtgcaggctg cctatcagaa ggtggtggct
     4021 ggtgtggcca atgccctggc tcacaaatac cactgagatc gatctttttc cctctgccaa
     4081 aaattatggg gacatcatga agccccttga gcatctgact tctggctaat aaaggaaatt
     4141 tattttcatt gcaatagtgt gttggaattt tttgtgtctc tcactcggaa ggacatatgg
     4201 gagggcaaat catttaaaac atcagaatga gtatttggtt tagagtttgg caacatatgc
     4261 ccatatgtaa ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct
     4321 gaaagcatgc ggaggaaatt ctccttgaag tttccctggt gttcaaagta aaggagtttg
     4381 caccagacgc acctctgttc actggtccgg cgtattaaaa cacgatacat tgttattagt
     4441 acatttatta agcgctagat tctgtgcgtt gttgatttac agacaattgt tgtacgtatt
     4501 ttaataattc attaaattta taatctttag ggtggtatgt tagagcgaaa atcaaatgat
     4561 tttcagcgtc tttatatctg aatttaaata ttaaatcctc aatagatttg taaaataggt
     4621 ttcgattagt ttcaaacaag ggttgttttt ccgaaccgat ggctggacta tctaatggat
     4681 tttcgctcaa cgccacaaaa cttgccaaat cttgtagcag caatctagct ttgtcgatat
     4741 tcgtttgtgt tttgttttgt aataaaggtt cgacgtcgtt caaaatatta tgcgcttttg
     4801 tatttctttc atcactgtcg ttagtgtaca attgactcga cgtaaacacg ttaaatagag
     4861 cttggacata tttaacatcg ggcgtgttag ctttattagg ccgattatcg tcgtcgtccc
     4921 aaccctcgtc gttagaagtt gcttccgaag acgattttgc catagccaca cgacgcctat
     4981 taattgtgtc ggctaacacg tccgcgatca aatttgtagt tgagcttttt ggaattgcga
     5041 tcgcataact tcgtatagca tacattatac gaagttataa gctcggaacg ctgcgctcgg
     5101 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
     5161 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
     5221 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg ccccctgacg agcatcacaa
     5281 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     5341 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     5401 gtccgccttt ctcccttcgg gaagcgtggc gctttctcaa tgctcacgct gtaggtatct
     5461 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     5521 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     5581 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     5641 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     5701 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     5761 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     5821 aaaaggatct caagaagatc ctttgttacc aatgcttaat cagtgaggca cctatctcag
     5881 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5941 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6001 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6061 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6121 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6181 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6241 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6301 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6361 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6421 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6481 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6541 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6601 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6661 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6721 aatattattg aagcatttat cagggttatt gtctcatgtc cgcgcgtt
//
