LOCUS       Exported                6497 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Green fluorescent probe for cGMP.
ACCESSION   .
VERSION     .
KEYWORDS    Green cGull
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6497)
  AUTHORS   Matsuda S, Harada K, Ito M, Takizawa M, Wongso D, Tsuboi T, 
            Kitaguchi T
  TITLE     Generation of a cGMP Indicator with an Expanded Dynamic Range by 
            Optimization of Amino Acid Linkers between a Fluorescent Protein and
            PDE5alpha.
  JOURNAL   ACS Sens. 2017 Jan 27;2(1):46-51. doi: 10.1021/acssensors.6b00582. 
            Epub 2016 Dec 29.
  PUBMED    28722423
REFERENCE   2  (bases 1 to 6497)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6497
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     94..113
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     347..364
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    481..502
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        517..547
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    555..571
                     /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)."
     primer_bind     560..582
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     579..595
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     579..595
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     polyA_signal    644..765
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(655..674)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     709..728
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(939..1733)
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(1050..1069)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     1660..1679
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(1800..2129)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      1814..1949
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     complement(1868..1887)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     primer_bind     2114..2134
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      complement(2143..2571)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2253..2274)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2465..2484
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     polyA_signal    2617..2841
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     2830..2847
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(2881..3132)
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNH
                     YLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     primer_bind     complement(2923..2944)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3683..3702
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3942..3963
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4053..4072)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4054..4072)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(4117..4320)
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     complement(4146..4166)
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     enhancer        4321..4700
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     primer_bind     4872..4891
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        4966..5070
                     /gene="bla"
                     /label=AmpR promoter
     CDS             5071..5931
                     /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     complement(5289..5308)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      6102..193
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
       61 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
      121 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
      181 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
      241 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
      301 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
      361 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
      421 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
      481 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
      541 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
      601 ttacgccaag ctctagctag aggtcgacgg tatacagaca tgataagata cattgatgag
      661 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
      721 gctattgctt tatttgtaac cattataagc tgcaataaac aagttggggt gggcgaagaa
      781 ctccagcatg agatccccgc gctggaggat catccagccg gcgtcccgga aaacgattcc
      841 gaagcccaac ctttcataga aggcggcggt ggaatcgaaa tctcgtgatg gcaggttggg
      901 cgtcgcttgg tcggtcattt cgaaccccag agtcccgctc agaagaactc gtcaagaagg
      961 cgatagaagg cgatgcgctg cgaatcggga gcggcgatac cgtaaagcac gaggaagcgg
     1021 tcagcccatt cgccgccaag ctcttcagca atatcacggg tagccaacgc tatgtcctga
     1081 tagcggtccg ccacacccag ccggccacag tcgatgaatc cagaaaagcg gccattttcc
     1141 accatgatat tcggcaagca ggcatcgcca tgggtcacga cgagatcctc gccgtcgggc
     1201 atgcgcgcct tgagcctggc gaacagttcg gctggcgcga gcccctgatg ctcttcgtcc
     1261 agatcatcct gatcgacaag accggcttcc atccgagtac gtgctcgctc gatgcgatgt
     1321 ttcgcttggt ggtcgaatgg gcaggtagcc ggatcaagcg tatgcagccg ccgcattgca
     1381 tcagccatga tggatacttt ctcggcagga gcaaggtgag atgacaggag atcctgcccc
     1441 ggcacttcgc ccaatagcag ccagtccctt cccgcttcag tgacaacgtc gagcacagct
     1501 gcgcaaggaa cgcccgtcgt ggccagccac gatagccgcg ctgcctcgtc ctgcagttca
     1561 ttcagggcac cggacaggtc ggtcttgaca aaaagaaccg ggcgcccctg cgctgacagc
     1621 cggaacacgg cggcatcaga gcagccgatt gtctgttgtg cccagtcata gccgaatagc
     1681 ctctccaccc aagcggccgg agaacctgcg tgcaatccat cttgttcaat catgcgaaac
     1741 gatcctcatc ctgtctcttg atcagatccg aaaatggata tacaagctcc cgggagcttt
     1801 ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact tctggaatag ctcagaggca
     1861 gaggcggcct cggcctctgc ataaataaaa aaaattagtc agccatgggg cggagaatgg
     1921 gcggaactgg gcggagttag gggcgggatg ggcggagtta ggggcgggac tatggttgct
     1981 gactaattga gatgcatgct ttgcatactt ctgcctgctg gggagcctgg ggactttcca
     2041 cacctggttg ctgactaatt gagatgcatg ctttgcatac ttctgcctgc tggggagcct
     2101 ggggactttc cacaccctaa ctgacacaca ttccacagaa ttaattcgcg ttaaattttt
     2161 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     2221 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     2281 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     2341 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     2401 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     2461 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     2521 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tggggatacc
     2581 ccctagagcc ccagctggtt ctttccgcct cagaagccat agagcccacc gcatccccag
     2641 catgcctgct attgtcttcc caatcctccc ccttgctgtc ctgccccacc ccacccccca
     2701 gaatagaatg acacctactc agacaatgcg atgcaatttc ctcattttat taggaaagga
     2761 cagtgggagt ggcaccttcc agggtcaagg aaggcacggg ggaggggcaa acaacagatg
     2821 gctggcaact agaaggcaca gtcgaggctg atcagcggtt taaacttaag cttcgaatta
     2881 cttgtacagc tcgtccatgc cgagagtgat cccggcggcg gtcacgaact ccagcaggac
     2941 catgtgatcg cgcttctcgt tggggtcttt gctcagggcg gactggtagc tcaggtagtg
     3001 gttgtcgggc agcagcacgg ggccgtcgcc gatgggggtg ttctgctggt agtggtcggc
     3061 gagctgcacg ctgccgtcct cgatgttgtg gcggatcttg aagttcacct tgatgccgtt
     3121 cttctgcttg tcggccatga tatagacgtt gtgcgtaaaa acattcacca agtaggccgc
     3181 aaagtccttt tcatcttttt cagtgaaggt cccaccattt cctgatttct tgttgatggc
     3241 ctgagctaca ccaacaacct cttccctgtg gttcttaatt ggcatacaaa ggatactttg
     3301 tgtcttgtag cctgtaattt ggtcaacttc tgcattgaac cgggggtcct cataggcatc
     3361 tttgatgttc aagggctcgc caaaagctgc cacatgtccc acaatgcctt tgttccactc
     3421 taaacggata cagttatttg aggcttcctc caatgttgaa ccttcagcaa catcaaagag
     3481 gcggctgatg aggaatttgt ctttagggct gtcctcacag acaaggaaca gggaatagcg
     3541 atccgcagag atgagtccat gcactccgcg catagagtac tccagcttgt gccccaggat
     3601 gttgccgtcc tccttgaagt cgatgccctt cagctcgatg cggttcacca gggtgtcgcc
     3661 ctcgaacttc acctcggcgc gggtcttgta gttgccgtcg tccttgaaga agatggtgcg
     3721 ctcctggacg tagccttcgg gcatggcgga cttgaagaag tcgtgctgct tcatgtggtc
     3781 ggggtagcgg gcgaagcaca tcaggccgta gccgaaggtg gtcacgaggg tgggccaggg
     3841 cacgggcagc ttgccggtgg tgcagatgaa cttcagggtc agcttgccgt aggtggcatc
     3901 gccctcgccc tcgccggaca cgctgaactt gtggccgttt acgtcgccgt ccagctcgac
     3961 caggatgggc accaccccgg tgaacagctc ctcgcccttg ctcaccatct cgagtctaga
     4021 gggcccgttt aaacgctagc cagcttgggt ctccctatag tgagtcgtat taatttcgat
     4081 aagccagtaa gcagtgggtt ctctagttag ccagagagct ctgcttatat agacctccca
     4141 ccgtacacgc ctaccgccca tttgcgtcaa tggggcggag ttgttacgac attttggaaa
     4201 gtcccgttga ttttggtgcc aaaacaaact cccattgacg tcaatggggt ggagacttgg
     4261 aaatccccgt gagtcaaacc gctatccacg cccattgatg tactgccaaa accgcatcac
     4321 catggtaata gcgatgacta atacgtagat gtactgccaa gtaggaaagt cccataaggt
     4381 catgtactgg gcataatgcc aggcgggcca tttaccgtca ttgacgtcaa tagggggcgt
     4441 acttggcata tgatacactt gatgtactgc caagtgggca gtttaccgta aatactccac
     4501 ccattgacgt caatggaaag tccctattgg cgttactatg ggaacatacg tcattattga
     4561 cgtcaatggg cgggggtcgt tgggcggtca gccaggcggg ccatttaccg taagttatgt
     4621 aacgcggaac tccatatatg ggctatgaac taatgacccc gtaattgatt actattaata
     4681 actagtcaat aatcaatgtc aacgcgtata tctggcccgt acatcgcgaa gcagcgcaaa
     4741 acgcctaacc ctaagcagat tcttcatgca attgtcggtc aagccttgcc ttgttgtagc
     4801 ttaaattttg ctcgcgcact actcagcgac ctccaacaca caagcaggga gcagatactg
     4861 gcttaactat gcggcatcag agcagattgt actgagagtg caccataggg gatcgggaga
     4921 tctcccgatc cgtcgacgtc aggtggcact tttcggggaa atgtgcgcgg aacccctatt
     4981 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa
     5041 atgcttcaat aatattgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt
     5101 attccctttt ttgcggcatt ttgccttcct gtttttgctc acccagaaac gctggtgaaa
     5161 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac
     5221 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt
     5281 aaagttctgc tatgtggcgc ggtattatcc cgtattgacg ccgggcaaga gcaactcggt
     5341 cgccgcatac actattctca gaatgacttg gttgagtact caccagtcac agaaaagcat
     5401 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac
     5461 actgcggcca acttacttct gacaacgatc ggaggaccga aggagctaac cgcttttttg
     5521 cacaacatgg gggatcatgt aactcgcctt gatcgttggg aaccggagct gaatgaagcc
     5581 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggcaacaac gttgcgcaaa
     5641 ctattaactg gcgaactact tactctagct tcccggcaac aattaataga ctggatggag
     5701 gcggataaag ttgcaggacc acttctgcgc tcggcccttc cggctggctg gtttattgct
     5761 gataaatctg gagccggtga gcgtgggtct cgcggtatca ttgcagcact ggggccagat
     5821 ggtaagccct cccgtatcgt agttatctac acgacgggga gtcaggcaac tatggatgaa
     5881 cgaaatagac agatcgctga gataggtgcc tcactgatta agcattggta actgtcagac
     5941 caagtttact catatatact ttagattgat ttaaaacttc atttttaatt taaaaggatc
     6001 taggtgaaga tcctttttga taatctcatg accaaaatcc cttaacgtga gttttcgttc
     6061 cactgagcgt cagaccccgt agaaaagatc aaaggatctt cttgagatcc tttttttctg
     6121 cgcgtaatct gctgcttgca aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg
     6181 gatcaagagc taccaactct ttttccgaag gtaactggct tcagcagagc gcagatacca
     6241 aatactgttc ttctagtgta gccgtagtta ggccaccact tcaagaactc tgtagcaccg
     6301 cctacatacc tcgctctgct aatcctgtta ccagtggctg ctgccagtgg cgataagtcg
     6361 tgtcttaccg ggttggactc aagacgatag ttaccggata aggcgcagcg gtcgggctga
     6421 acggggggtt cgtgcacaca gcccagcttg gagcgaacga cctacaccga actgagatac
     6481 ctacagcgtg agctatg
//
