LOCUS       Exported                6091 bp ds-DNA     circular SYN 25-JUL-2025
DEFINITION  Fluorescent reporter for glutamate, third generation, variant 857. 
            iGluSnFR3.v857.
ACCESSION   .
VERSION     .
KEYWORDS    pAAV.hSyn.FLEX.iGluSnFR3.v857.PDGFR.codonopt
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6091)
  TITLE     A third-generation glutamate indicator optimized for synapses
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6091)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Thursday, Jul 31, 2025 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6091
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          4594..4635
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          4636..5076
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      69..524
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(156..175)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     366..387
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        550..654
                     /gene="bla"
                     /label=AmpR promoter
     CDS             655..1515
                     /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(873..892)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1686..2274
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2175..2194
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2428..2445
                     /label=L4440
                     /note="L4440 vector, forward primer"
     repeat_region   2511..2640
                     /label=AAV2 ITR
     primer_bind     complement(2522..2533)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     2583..2594
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    2802..2923
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2839..2858)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2893..2912
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     misc_feature    3001..3589
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3107..3118
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     3516..3536
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     protein_bind    complement(3661..3694)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    complement(3765..3798)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     regulatory      3823..3828
                     /regulatory_class="other"
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             3829..3840
                     /codon_start=1
                     /label=MCS
                     /note="MCS"
                     /translation="MGRS"
     CDS             3841..4116
                     /codon_start=1
                     /label=cpSFGFP 147-238
                     /note="cpSFGFP 147-238"
                     /translation="SHNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDG
                     PVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     misc_feature    3859..3861
                     /label=T153M
                     /note="T153M"
     misc_feature    3874..3876
                     /label=R158K
                     /note="R158K"
     misc_feature    3889..3891
                     /label=V163A (sfGFP)
                     /note="V163A (sfGFP)"
     misc_feature    3913..3915
                     /label=I171V (sfGFP)
                     /note="I171V (sfGFP)"
     misc_feature    3925..3927
                     /label=G175S
                     /note="G175S (175S in sfGFP, 175G in Venus. Keeping 175S.) 
                     "
     misc_feature    3940..3942
                     /label=Y180D - spontaneous mutation carried
                     /note="Y180D - spontaneous mutation carried over from 
                     SF-iGluSnFR. Surface mutation, not expected to affect 
                     Fluorescence."
     misc_feature    4018..4020
                     /label=A206V (sfGFP)
                     /note="A206V (sfGFP)"
     misc_feature    4024..4026
                     /label=Silent codon change
                     /note="Silent codon change"
     CDS             4117..4134
                     /codon_start=1
                     /label=Linker
                     /note="Linker"
                     /translation="GGTGGS"
     CDS             4135..4572
                     /codon_start=1
                     /label=cpSFGFP 1-146
                     /note="cpSFGFP 1-146"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFN"
     misc_feature    4222..4224
                     /label=S30R (sfGFP)
                     /note="S30R (sfGFP)"
     misc_feature    4249..4251
                     /label=Y39N (sfGFP)
                     /note="Y39N (sfGFP)"
     misc_feature    4270..4272
                     /label=F46L Venus fast maturation
                     /note="F46L Venus fast maturation"
     misc_feature    4327..4329
                     /label=TYG Chromophore
                     /note="GYG Chromophore for Venus"
     misc_feature    4336..4338
                     /label=V68L in Venus. (Keeping V from sfGFP)
                     /note="V68L in Venus. (Keeping V from sfGFP)"
     misc_feature    4348..4350
                     /label=S72
                     /note="S72A"
     misc_feature    4411..4413
                     /label=I93V (sfGFP)
                     /note="I93V (sfGFP)"
     misc_feature    4429..4431
                     /label=F99S (sfGFP)
                     /note="F99S (sfGFP)"
     misc_feature    4447..4449
                     /label=N105T (sfGFP)
                     /note="N105T (sfGFP)"
     CDS             4573..4578
                     /codon_start=1
                     /label=MCS
                     /translation="LQ"
     CDS             4579..4593
                     /codon_start=1
                     /label=linker
                     /note="linker"
                     /translation="STGSG"
     CDS             4594..4635
                     /codon_start=1
                     /label=3xGGGGS
                     /note="/__SeqFeature__=True"
                     /note="/__level__=0"
                     /note="/application_notes=Commonly used flexible linker 
                     forfusing two proteins."
                     /note="/full_name=3 tandem GGGGS"
                     /note="/name=3xGGGGS"
                     /note="/official_designation=3xGGGGS"
                     /note="/uuid=82e342ab8d8e4d71a1f1c7026d54944d"
                     /note="/vntifkey=21"
                     /translation="GGGTGGGGSGGGGS"
     CDS             4636..5076
                     /codon_start=1
                     /label=miRFP670nano3
                     /translation="MANLDKMLNTTVTEVRKFLQADRVCVFKFEEDYSGTVSHEAVDDR
                     WISILKTQVQDRYFMETRGEEYVHGRYQAIADIYTANLVECYRDLLIEFQVRAILAVPI
                     LQGKKLWGLLVAHQLAGPREWQTWEIDFLKQQAVVMGIAIQQS"
     protein_bind    5092..5125
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     protein_bind    5188..5221
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     primer_bind     complement(5232..5251)
                     /label=pMT2-F
                     /note="Synthetic intron, forward primer"
     intron          5234..5366
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     repeat_region   5959..6088
                     /label=AAV2 ITR
     primer_bind     complement(6005..6016)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     primer_bind     6066..6077
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa cagttgcgca gcctgaatgg
       61 cgaatgggac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
      121 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
      181 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
      241 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
      301 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
      361 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
      421 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
      481 aaaatttaac gcgaatttta acaaaatatt aacgcttaca atttaggtgg cacttttcgg
      541 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
      601 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
      661 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
      721 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
      781 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
      841 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt
      901 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
      961 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     1021 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     1081 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     1141 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgta
     1201 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     1261 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     1321 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     1381 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     1441 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     1501 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     1561 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     1621 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     1681 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     1741 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     1801 ggcttcagca gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac
     1861 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     1921 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     1981 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     2041 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     2101 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     2161 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     2221 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     2281 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     2341 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     2401 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     2461 ccaatacgca aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctgcgcgctc
     2521 gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt ggtcgcccgg
     2581 cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact aggggttcct
     2641 tgtagttaat gattaacccg ccatgctact tatctacgta gccatgctct aggaagatcc
     2701 ttatcgattt taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacatctcc
     2761 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     2821 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     2881 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgctcgaagc
     2941 ggccgcccgg gtcgactcta gatgcatgct cgagcggccg cccgggtcga cggtatcgat
     3001 gcggggaggc ggcccaaagg gagatccgac tcgtctgagg gcgaaggcga agacgcggaa
     3061 gaggccgcag agccggcagc aggccgcggg aaggaaggtc cgctggattg agggccgaag
     3121 ggacgtagca gaaggacgtc ccgcgcagaa tccaggtggc aacacaggcg agcagccaag
     3181 gaaaggacga tgatttcccc gacaacacca cggaattgtc agtgcccaac agccgagccc
     3241 ctgtccagca gcgggcaagg caggcggcga tgagttccgc cgtggcaata gggaggggga
     3301 aagcgaaagt cccggaaagg agctgacagg tggtggcaat gccccaacca gtgggggttg
     3361 cgtcagcaaa cacagtgcac accacgccac gttgcctgac aacgggccac aactcctcat
     3421 aaagagacag caaccaggat ttatacaagg aggagaaaat gaaagccata cgggaagcaa
     3481 tagcatgata caaaggcatt aaagcagcgt atccacatag cgtaaaagga gcaacatagt
     3541 taagaatacc agtcaatctt tcacaaattt tgtaatccag aggttgatta tcgataagct
     3601 tctcgagtgg atcatctgca gaattcgccc ttttcagtgt ttcgaacctc ttcgagggac
     3661 ataacttcgt ataatgtatg ctatacgaag ttatgggtcg atggtgagat ctggactaga
     3721 gggtcgatgg tgatgcttgg atcaaaatag gaagaatccc aatgataact tcgtataaag
     3781 tatcctatac gaagttatac tagtggtacc accggtgata tcgccaccat gggcagatct
     3841 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggc gaacttcaag
     3901 atccgccaca acgtggagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     3961 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgtg
     4021 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     4081 gccgggatca ctctcggcat ggacgagctg tacaagggcg gtaccggagg gagcatgagc
     4141 aagggcgagg agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta
     4201 aacggccaca agttcagcgt gcgcggcgag ggcgagggcg atgccaccaa cggcaagctg
     4261 accctgaagt tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc
     4321 accctgacct acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac
     4381 ttcttcaagt ccgccatgcc cgaaggctac gtccaggagc gcaccatcag cttcaaggac
     4441 gacggcacct acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc
     4501 atcgagctga agggcatcga cttcaaggag gacggcaaca tcctggggca taagctggag
     4561 tacaacttta acctgcagtc taccggctcc ggaggtggag gtactggagg aggtgggtca
     4621 ggtggaggtg gatcaatggc aaacctggac aagatgctga acaccaccgt gaccgaggtg
     4681 cgcaagttcc tgcaagcgga cagagtgtgc gtgttcaagt tcgaggaaga ttactccggc
     4741 accgtctcgc acgaagccgt ggacgacaga tggattagca tcctgaagac ccaggtgcag
     4801 gacagatact tcatggaaac cagaggcgag gaatacgtcc acggcagata ccaggccatc
     4861 gccgacatct acacagccaa tctggtcgag tgctacagag acctgctgat cgagtttcag
     4921 gtgcgggcca ttctggctgt ccccatcctg caaggcaaga agctgtgggg cctgctggtg
     4981 gcccaccaac tggccggccc tcgggagtgg cagacctggg aaatcgactt cctgaaacag
     5041 caagccgtgg tgatgggcat cgccatccag cagagctagg ctagcactag tataacttcg
     5101 tatagcatac attatacgaa gttatgtgtc gatggtgatg cttggcaatt cgggtcgatg
     5161 gtgaagcatt ggtcttccta ttttgatata acttcgtata ggatacttta tacgaagtta
     5221 tggggatcat cacctgtgga gagaaaggca aagtggatgt cagtaagacc aataggtgcc
     5281 tatcagaaac gcaagagtct tctctgtctc gacaagccca gtttctattg gtctccttaa
     5341 acctgtcttg taaccttgat acttacctga tccaacggct gcgacttggg gcagggggtc
     5401 ctaggggtgg tctggccagg agccgcgggg gcggactgcg cggtgcccag gagcacagct
     5461 gcgctctcag gcacgacacg actcctccgc tgcccaccgc agactgaggc agcgctgagt
     5521 cgccggcgcc gcagcgcaga tggtcgcgcc cgtgcccccc tatctcgcgc ctcgcgtggt
     5581 gcggtccggc tgggccggcg gcggcgcgga cgcgaccaag gtggccggga aggggagttt
     5641 gcgggggacc ggcgagtgac gtcagcgcgc cttcagtgct gaggcggcgg tggcgcgcgc
     5701 cgccaggcgg gggcgaaggc actgtccgcg gtgctgaagc tggcagtgcg cacgcgcctc
     5761 gccgcatcct gtttcccctc cccctctctg ataggggatg cgcaatttgg ggaatggggg
     5821 ttgggtgctt gtccagtggg tcggggtcgg tcgtcaattt aaatactgaa aagggcgaat
     5881 tccagcacac tggagctcgg atcttcctag agcatggcta cgtagataag tagcatggcg
     5941 ggttaatcat taactacaag gaacccctag tgatggagtt ggccactccc tctctgcgcg
     6001 ctcgctcgct cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg
     6061 cggcctcagt gagcgagcga gcgcgcagct g
//
