LOCUS       Exported                6422 bp ds-DNA     circular SYN 16-MAR-2023
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6422)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Monday, Sep 25, 2023 from SnapGene 5.0.8
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6422
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     source          4596..5528
     misc_feature    complement(23..187)
                     /label=ITR
                     /note="ITR"
     misc_feature    complement(194..236)
                     /label=seq
                     /note="seq"
     misc_feature    complement(251..270)
                     /label=pzac2.1 5088f
                     /note="pzac2.1 5088f"
     primer_bind     complement(351..374)
                     /label=f.ITR for 2.01.14
                     /note="f.ITR for 2.01.14"
     primer_bind     complement(452..472)
                     /label=f.ITR for 1.01.13
                     /note="f.ITR for 1.01.13"
     primer_bind     2471..2489
                     /label=r.ITR rev 3.01.19
                     /note="r.ITR rev 3.01.19"
     primer_bind     2513..2536
                     /label=r.ITR rev 4.01.20
                     /note="r.ITR rev 4.01.20"
     misc_feature    complement(2707..2871)
                     /label=ITR
                     /note="ITR"
     promoter        2898..3832
                     /label=CAG
                     /note="CAG "
     primer_bind     2975..2994
                     /label=f.CAG promoter seq.33.1743
                     /note="f.CAG promoter seq.33.1743"
     primer_bind     complement(3043..3058)
                     /label=r.CAG-flex ITR seq.33.1306 CAG rev
                     /note="r.CAG-flex ITR seq.33.1306 CAG rev"
     primer_bind     3049..3066
                     /label=f.CAG promoter.33.1744
                     /note="f.CAG promoter.33.1744"
     primer_bind     3483..3499
                     /label=f.CAG promoter seq.33.1747
                     /note="f.CAG promoter seq.33.1747"
     primer_bind     3495..3516
                     /label=r.CAG promoter seq.33.1745
                     /note="r.CAG promoter seq.33.1745"
     primer_bind     complement(3581..3603)
                     /label=r.CAG promoter seq.33.1746
                     /note="r.CAG promoter seq.33.1746"
     primer_bind     3703..3725
                     /label=f.CAG for.01.40
                     /note="f.CAG for.01.40"
     primer_bind     complement(3795..3818)
                     /label=r.CAG promoter seq.33.1748
                     /note="r.CAG promoter seq.33.1748"
     misc_feature    3833
                     /label=pCAG_GCaMP3
     misc_feature    3840..3845
                     /label=Kozak
     CDS             3846..3857
                     /codon_start=1
                     /label=MCS
                     /note="MCS"
                     /translation="MGRS"
     CDS             3858..4133
                     /codon_start=1
                     /label=cpSFGFP 147-238
                     /note="cpSFGFP 147-238"
                     /translation="SHNVYIMADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDG
                     PVLLPDNHYLSTQSVLSKDPNEKRDHMVLLEFVTAAGITLGMDELYK"
     misc_feature    3876..3878
                     /label=T153M
                     /note="T153M"
     misc_feature    3891..3893
                     /label=R158K
                     /note="R158K"
     misc_feature    3906..3908
                     /label=V163A (sfGFP)
                     /note="V163A (sfGFP)"
     misc_feature    3930..3932
                     /label=I171V (sfGFP)
                     /note="I171V (sfGFP)"
     misc_feature    3942..3944
                     /label=G175S
                     /note="G175S (175S in sfGFP, 175G in Venus. Keeping 175S.) 
                     "
     misc_feature    3957..3959
                     /label=Y180D - spontaneous mutation carried
                     /note="Y180D - spontaneous mutation carried over from 
                     SF-iGluSnFR. Surface mutation, not expected to affect 
                     Fluorescence."
     misc_feature    4035..4037
                     /label=A206V (sfGFP)
                     /note="A206V (sfGFP)"
     misc_feature    4041..4043
                     /label=Silent codon change
                     /note="Silent codon change"
     CDS             4134..4151
                     /codon_start=1
                     /label=Linker
                     /note="Linker"
                     /translation="GGTGGS"
     CDS             4152..4589
                     /codon_start=1
                     /label=cpSFGFP 1-146
                     /note="cpSFGFP 1-146"
                     /translation="MSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDDG
                     TYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFN"
     misc_feature    4239..4241
                     /label=S30R (sfGFP)
                     /note="S30R (sfGFP)"
     misc_feature    4266..4268
                     /label=Y39N (sfGFP)
                     /note="Y39N (sfGFP)"
     misc_feature    4287..4289
                     /label=F46L Venus fast maturation
                     /note="F46L Venus fast maturation"
     misc_feature    4344..4346
                     /label=TYG Chromophore
                     /note="GYG Chromophore for Venus"
     misc_feature    4353..4355
                     /label=V68L in Venus. (Keeping V from sfGFP)
                     /note="V68L in Venus. (Keeping V from sfGFP)"
     misc_feature    4365..4367
                     /label=S72
                     /note="S72A"
     misc_feature    4428..4430
                     /label=I93V (sfGFP)
                     /note="I93V (sfGFP)"
     misc_feature    4446..4448
                     /label=F99S (sfGFP)
                     /note="F99S (sfGFP)"
     misc_feature    4464..4466
                     /label=N105T (sfGFP)
                     /note="N105T (sfGFP)"
     CDS             4590..4595
                     /codon_start=1
                     /label=MCS
                     /translation="LQ"
     CDS             4596..4637
                     /codon_start=1
                     /label=3xGGGGS
                     /note="/__SeqFeature__=True"
                     /note="/__level__=0"
                     /note="/application_notes=Commonly used flexible linker for
                     fusing two proteins."
                     /note="/full_name=3 tandem GGGGS"
                     /note="/name=3xGGGGS"
                     /note="/official_designation=3xGGGGS"
                     /note="/uuid=82e342ab8d8e4d71a1f1c7026d54944d"
                     /note="/vntifkey=21"
                     /translation="GGGSGGGGSGGGGS"
     CDS             4638..5528
                     /codon_start=1
                     /label=HaloTag
                     /label=Halo tag/SV40 NLS
                     /note="/__SeqFeature__=True"
                     /note="/__level__=0"
                     /note="/name=Halo tag/SV40 NLS"
                     /note="/official_designation=Halo tag/SV40 NLS"
                     /note="/uuid=3b230e3a699845e5874505cf15bc074d"
                     /note="/vntifkey=21"
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRTIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNVKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     primer_bind     5394..5403
                     /label=f.CAG promoter.33.1744
                     /note="f.CAG promoter.33.1744"
     CDS             5529..5546
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     misc_feature    5569..6157
                     /label=WPRE
                     /note="WPRE"
     primer_bind     complement(5640..5659)
                     /label=r.wpre rev.01.3
                     /note="r.wpre rev.01.3"
     primer_bind     complement(5970..5990)
                     /label=wpre rev pcr 32.63
                     /note="wpre rev pcr 32.63"
     primer_bind     5994..6013
                     /label=f.WPRE seq.01.80
                     /note="f.WPRE seq.01.80"
     primer_bind     6201..6224
                     /label=f.ITR for 3.01.15
                     /note="f.ITR for 3.01.15"
ORIGIN
        1 ggatcttcct agagcatggc tacgtagata agtagcatgg cgggttaatc attaactaca
       61 aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg
      121 ccgggcgacc aaaggtcgcc cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc
      181 gagcgcgcag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg
      241 gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc
      301 ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg
      361 aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct
      421 ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca
      481 gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct
      541 cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc
      601 gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt
      661 tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc
      721 cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc
      781 cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg
      841 gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc tgctgaagcc
      901 agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag
      961 cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga
     1021 tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat
     1081 tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag
     1141 ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat
     1201 cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc
     1261 cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat
     1321 accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag
     1381 ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg
     1441 ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc
     1501 tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca
     1561 acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg
     1621 tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc
     1681 actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta
     1741 ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc
     1801 aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg
     1861 ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc
     1921 cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc
     1981 aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat
     2041 actcatactc ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag
     2101 cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc
     2161 ccgaaaagtg ccacctaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt
     2221 tgttaaatca gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca
     2281 aaagaataga ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta
     2341 aagaacgtgg actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta
     2401 cgtgaaccat caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg
     2461 aaccctaaag ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga
     2521 aaggaaggga agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg
     2581 ctgcgcgtaa ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg
     2641 ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc gggcctcttc gctattacgc
     2701 cagctgcgcg ctcgctcgct cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc
     2761 tttggtcgcc cggcctcagt gagcgagcga gcgcgcagag agggagtggc caactccatc
     2821 actaggggtt ccttgtagtt aatgattaac ccgccatgct acttatctac gtagccatgc
     2881 tctaggaaga tcgtaccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata
     2941 gggactttcc attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta
     3001 catcaagtgt atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc
     3061 gcctggcatt atgcccagta catgacctta tgggactttc ctacttggca gtacatctac
     3121 gtattagtca tcgctattac catggtcgag gtgagcccca cgttctgctt cactctcccc
     3181 atctcccccc cctccccacc cccaattttg tatttattta ttttttaatt attttgtgca
     3241 gcgatggggg cggggggggg gggggggcgc gcgccaggcg gggcggggcg gggcgagggg
     3301 cggggcgggg cgaggcggag aggtgcggcg gcagccaatc agagcggcgc gctccgaaag
     3361 tttcctttta tggcgaggcg gcggcggcgg cggccctata aaaagcgaag cgcgcggcgg
     3421 gcgggagtcg ctgcgacgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
     3481 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
     3541 tcctccgggc tgtaattagc gcttggttta atgacggctt gtttcttttc tgtggctgcg
     3601 tgaaagcctt gaggggctcc gggagggccc tttgtgcggg gggagcggct cggggctgtc
     3661 cgcgggggga cggctgcctt cgggggggac ggggcagggc ggggttcggc ttctggcgtg
     3721 tgaccggcgg ctctagagcc tctgctaacc atgttcatgc cttcttcttt ttcctacagc
     3781 tcctgggcaa cgtgctggtt attgtgctgt ctcatcattt tggcaaagaa ttggatcccg
     3841 ccaccatggg cagatctagc cacaacgtct atatcatggc cgacaagcag aagaacggca
     3901 tcaaggcgaa cttcaagatc cgccacaacg tggaggacgg cagcgtgcag ctcgccgacc
     3961 actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac aaccactacc
     4021 tgagcaccca gtccgtgctg agcaaagacc ccaacgagaa gcgcgatcac atggtcctgc
     4081 tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac aagggcggta
     4141 ccggagggag catgagcaag ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg
     4201 agctggacgg cgacgtaaac ggccacaagt tcagcgtgcg cggcgagggc gagggcgatg
     4261 ccaccaacgg caagctgacc ctgaagttca tctgcaccac cggcaagctg cccgtgccct
     4321 ggcccaccct cgtgaccacc ctgacctacg gcgtgcagtg cttcagccgc taccccgacc
     4381 acatgaagca gcacgacttc ttcaagtccg ccatgcccga aggctacgtc caggagcgca
     4441 ccatcagctt caaggacgac ggcacctaca agacccgcgc cgaggtgaag ttcgagggcg
     4501 acaccctggt gaaccgcatc gagctgaagg gcatcgactt caaggaggac ggcaacatcc
     4561 tggggcataa gctggagtac aactttaacc tgcagggcgg aggctcgggc ggaggtgggt
     4621 cgggtggcgg cggatcaatg gccgagatcg gcaccggctt ccccttcgac ccccactacg
     4681 tggaggtgct gggcgagcgc atgcactacg ttgacgtggg cccccgcgac ggcacccccg
     4741 tgctgttcct gcacggcaac cccaccagca gctacgtgtg gcgcaacatc atcccccacg
     4801 tggcccccac ccaccgcact atcgcccccg acctgatcgg catgggcaag agcgacaagc
     4861 ccgacctggg ctacttcttc gacgaccacg tgcgcttcat ggacgccttc atcgaggccc
     4921 tgggcctgga ggaggtggtg ctggtgatcc acgactgggg cagcgccctg ggcttccact
     4981 gggccaagcg caaccccgag cgcgtgaagg gcatcgcctt catggagttc atccgcccca
     5041 tccccacctg ggacgagtgg cccgagttcg cccgcgagac cttccaggcc ttccgcacca
     5101 ccgacgtggg ccgcaagctg atcatcgacc agaacgtgtt catcgagggc accctgccca
     5161 tgggcgtggt gcgccccctg accgaggtgg agatggacca ctaccgcgag cccttcctga
     5221 accccgtgga ccgcgagccc ctgtggcgct tccccaacga gctgcccatc gccggcgagc
     5281 ccgccaacat cgtggccctg gtggaggagt acatggactg gctgcaccag agccccgtgc
     5341 ccaagctgct gttctggggc acccccggcg tgctgatccc ccccgccgag gccgcccgcc
     5401 tggccaagag cctgcccaac gttaaggccg tggacatcgg ccccggcctg aacctgctcc
     5461 aggaggacaa ccccgacctg atcggcagcg agatcgcccg ctggctgagc accctcgaga
     5521 tcagcggcca tcaccatcac caccattaag aattcgaagc ttatcgataa tcaacctctg
     5581 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     5641 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     5701 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     5761 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     5821 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     5881 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     5941 aattccgtgg tgttgtcggg gaaatcatcg tcctttcctt ggctgctcgc ctgtgttgcc
     6001 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     6061 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     6121 cagacgagtc ggatctccct ttgggccgcc tccccgcatc gataccgtcg acccgggcgg
     6181 ccgcttcgag cagacatgat aagatacatt gatgagtttg gacaaaccac aactagaatg
     6241 cagtgaaaaa aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt
     6301 ataagctgca ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag
     6361 ggggagatgt gggaggtttt ttaaagcaag taaaacctct acaaatgtgg taaaatcgat
     6421 aa
//
