LOCUS       Exported                6563 bp ds-DNA     circular SYN 30-OCT-2019
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  recombinant plasmid
REFERENCE   1  (bases 1 to 6563)
  AUTHORS   Manuel Mohr
  TITLE     Direct Submission
  JOURNAL   Exported Wednesday, Oct 30, 2019 from SnapGene 5.0.1
            https://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6563
                     /organism="recombinant plasmid"
                     /mol_type="other DNA"
     rep_origin      complement(186..774)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     CDS             complement(945..1805)
                     /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"
     promoter        complement(1806..1910)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(1936..2391)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     2533..2549
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    complement(2568..2735)
                     /label=ITR
                     /note="ITR"
     polyA_signal    2859..2980
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     misc_feature    3058..3646
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     CDS             3164..3175
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     misc_feature    3556..3575
                     /label=WPRE rev WPRE rev ttaaagcagcgtatccacat
                     /note="WPRE rev WPRE rev ttaaagcagcgtatccacat"
     misc_feature    complement(3673..3678)
                     /label=Former PstI site
                     /note="Former PstI site"
     misc_feature    complement(3678..3683)
                     /label=Former EcoRI site
                     /note="Former EcoRI site"
     protein_bind    complement(3718..3751)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    complement(3765..3770)
                     /label=Former BglII site
                     /note="Former BglII site"
     protein_bind    complement(3822..3855)
                     /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."
     regulatory      3880..3888
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             3886..3888
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="M"
     CDS             3889..3945
                     /codon_start=1
                     /label=GAP43 Sequence
                     /note="GAP43 Sequence"
                     /translation="LCCMRRTKQVEKNDEDQKI"
     CDS             3946..4050
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="GSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMV"
     misc_feature    3946..4050
                     /label=PRSET Leader
                     /note="PRSET Leader"
     misc_feature    3952..3969
                     /label=6xHisTag
                     /note="6xHisTag"
     regulatory      4039..4048
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             4048..5293
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6s
                     /translation="VDSSRRKWNKTGHAVRVIGRLSSLENVYITADKQKNGIKANFHIR
                     HNIEDGGVQLAYHYQQNTPIGDGPVLLPDNHYLSYQPVLSKDPNEKRDHMVLLEFVTAA
                     GITLGMDELYKGGTGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGK
                     LTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFF
                     KDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKE
                     LFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMA
                     RKMKYRDTEEEIREAFGVFDKDGNGYISAAELRHVMTNLGEKLTDEEVDEMIREADIDG
                     DGQVNYEEFVQMMTAK"
     misc_difference 4096..4098
                     /label=A52V (GCaMP7s mutation)
     misc_difference 4135..4137
                     /label=K65T (K153T, sf-mutation, actually M in Citrine)
     misc_difference 4174..4176
                     /label=K78H (GCaMP7s mutation)
     misc_difference 4285..4287
                     /label=V115Y (V203Y, pi-stack muation for yel. Chromoph)
     misc_feature    4291..4293
                     /label=S117P (S205P,Kaspar's rexcue muatation)
     misc_difference 4294..4296
                     /label=K118V (K206V, close to rescue mutation)
     misc_difference 4549..4551
                     /label=F203L (F36L, maturation mutation in Venus)
     misc_feature    4606..4608
                     /label=T222G (T65G, yellow Chromophore mutation)
     misc_difference 4615..4617
                     /label=V225L (V68L, from Citrine seq, unknown effect)
     misc_difference 4627..4629
                     /label=S229A (S72A, yellow mutation, A in all YFPs)
     misc_difference 4690..4692
                     /label=I250V (I93V, unknown effect, V in all YFPs&sfGFP)
     misc_difference 4846..4848
                     /label=T302L (GCaMP7s mutation
     misc_difference 4849..4851
                     /label= R303P (GCaMP7s mutation)
     misc_difference 4891..4893
                     /label=A317L (GCaMP7s mutation)
     misc_difference 5080..5082
                     /label=D380Y (GCaMP7s mutation)
     misc_difference 5083..5085
                     /label=T381R (GCaMP7s mutation)
     misc_difference 5089..5091
                     /label=S383T (GCaMP7s mutation)
     misc_difference 5116..5118
                     /label=R392G (GCaMP7s mutation)
     protein_bind    5313..5346
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     misc_feature    complement(5348)
                     /label=C to A switch indicated in sequencing
                     /note="C to A switch indicated in sequencing"
     protein_bind    5409..5442
                     /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."
     misc_feature    5450..5469
                     /label=intron AscI 3'r
                     /note="intron AscI 3'r"
     intron          5455..5587
                     /label=chimeric intron
                     /note="chimera between introns from human beta-globin and 
                     immunoglobulin heavy chain genes"
     misc_feature    complement(5590..6077)
                     /label=synapsin promoter
                     /note="synapsin promoter"
     misc_feature    complement(6041..6059)
                     /label=syn forward primer cactggacaagcacccaac
                     /note="syn forward primer cactggacaagcacccaac"
     misc_feature    complement(6057..6077)
                     /label=Syn speI 5'f
                     /note="Syn speI 5'f"
     misc_feature    complement(6095..6113)
                     /label=AA0208  SacI 480 f
                     /note="AA0208  SacI 480 f"
     misc_feature    complement(6142..6309)
                     /label=ITR
                     /note="ITR"
     primer_bind     complement(6347..6363)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6371..6387
                     /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(6395..6425)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6440..6461
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
       61 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
      121 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
      181 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
      241 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
      301 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
      361 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
      421 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
      481 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
      541 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
      601 tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct gctgaagcca
      661 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
      721 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
      781 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
      841 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
      901 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
      961 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     1021 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     1081 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     1141 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     1201 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     1261 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     1321 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     1381 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     1441 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     1501 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     1561 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     1621 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     1681 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     1741 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     1801 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     1861 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     1921 cgaaaagtgc cacctaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt
     1981 gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa
     2041 aagaatagac cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa
     2101 agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac
     2161 gtgaaccatc accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga
     2221 accctaaagg gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa
     2281 aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc
     2341 tgcgcgtaac caccacaccc gccgcgctta atgcgccgct acagggcgcg tcccattcgc
     2401 cattcaggct gcgcaactgt tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc
     2461 agctggcgaa agggggatgt gctgcaaggc gattaagttg ggtaacgcca gggttttccc
     2521 agtcacgacg ttgtaaaacg acggccagtg aattaggtta attaaggctg cgcgctcgct
     2581 cgctcactga ggccgcccgg gcaaagcccg ggcgtcgggc gacctttggt cgcccggcct
     2641 cagtgagcga gcgagcgcgc agagagggag tggccaactc catcactagg ggttccttgt
     2701 agttaatgat taacccgcca tgctacttat ctacgtagcc atgctctagg aagatcctta
     2761 tcgattttac cacatttgta gaggttttac ttgctttaaa aaacctccca catctccccc
     2821 tgaacctgaa acataaaatg aatgcaattg ttgttgttaa cttgtttatt gcagcttata
     2881 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     2941 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgc tcgaagcggc
     3001 cgcccgggtc gactctagat gcatgctcga gcggccgccc gggtcgacgg tatcgatgcg
     3061 gggaggcggc ccaaagggag atccgactcg tctgagggcg aaggcgaaga cgcggaagag
     3121 gccgcagagc cggcagcagg ccgcgggaag gaaggtccgc tggattgagg gccgaaggga
     3181 cgtagcagaa ggacgtcccg cgcagaatcc aggtggcaac acaggcgagc agccaaggaa
     3241 aggacgatga tttccccgac aacaccacgg aattgtcagt gcccaacagc cgagcccctg
     3301 tccagcagcg ggcaaggcag gcggcgatga gttccgccgt ggcaataggg agggggaaag
     3361 cgaaagtccc ggaaaggagc tgacaggtgg tggcaatgcc ccaaccagtg ggggttgcgt
     3421 cagcaaacac agtgcacacc acgccacgtt gcctgacaac gggccacaac tcctcataaa
     3481 gagacagcaa ccaggattta tacaaggagg agaaaatgaa agccatacgg gaagcaatag
     3541 catgatacaa aggcattaaa gcagcgtatc cacatagcgt aaaaggagca acatagttaa
     3601 gaataccagt caatctttca caaattttgt aatccagagg ttgattatcg ataagcttct
     3661 cgagtggatc atttgcagaa ttggcccttt tcagtgtttc gaacctcttc gagggacata
     3721 acttcgtata atgtatgcta tacgaagtta tgggtcgatg gtgagatttg gactagaggg
     3781 tcgatggtga tgcttggatc aaaataggaa gaatcccaat gataacttcg tataaagtat
     3841 cctatacgaa gttatactag tggtaccacc ggtgatatcg ccaccatgct gtgctgtatg
     3901 agaagaacca aacaggttga aaagaatgat gaggaccaaa agattggttc tcatcatcat
     3961 catcatcatg gtatggctag catgactggt ggacagcaaa tgggtcggga tctgtacgac
     4021 gatgacgata aggatctcgc caccatggtc gactcatcac gtcgtaagtg gaataagaca
     4081 ggtcacgcag tcagagtgat aggtcggctg agctcactcg agaacgtcta tatcaccgcc
     4141 gacaagcaga agaacggcat caaggcgaac ttccacatcc gccacaacat cgaggacggc
     4201 ggcgtgcagc tcgcctatca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg
     4261 ctgcccgaca accactacct gagctaccag cccgtgctta gcaaagaccc caacgagaag
     4321 cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct gggcatggat
     4381 gagctgtaca agggcggtac cggagggagc atggtgagca agggcgagga gctgttcacc
     4441 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     4501 tccggcgagg gtgagggcga tgccacctac ggcaagctga ccctgaagct gatttgcacc
     4561 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgggcta cggcctgcag
     4621 tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     4681 gaaggctacg tgcaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     4741 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagcttaa gggcatcgac
     4801 ttcaaggagg acggcaacat cctggggcac aagctggagt acaaccttcc tgaccaactg
     4861 actgaagagc agatcgcaga atttaaagag cttttctccc tatttgacaa ggacggggat
     4921 gggacaataa caaccaagga gctggggacg gtgatgcggt ctctggggca gaaccccaca
     4981 gaagcagagc tgcaggacat gatcaatgaa gtagatgccg acggtgacgg cacaatcgac
     5041 ttccctgagt tcctgacaat gatggcaaga aaaatgaaat acagggacac ggaagaagaa
     5101 attagagaag cgttcggtgt gtttgataag gatggcaatg gctacatcag tgcagcagag
     5161 cttcgccacg tgatgacaaa ccttggagag aagttaacag atgaagaggt tgatgaaatg
     5221 atcagggaag cagacatcga tggggatggt caggtaaact acgaagagtt tgtacaaatg
     5281 atgacagcga agtaggatcc gctagcacta gtataacttc gtatagcata cattatacga
     5341 agttatgtgt cgatggtgat gcttggcaat tcgggtcgat ggtgaagcat tggtcttcct
     5401 attttgatat aacttcgtat aggatacttt atacgaagtt atggggatca tcacctgtgg
     5461 agagaaaggc aaagtggatg tcagtaagac caataggtgc ctatcagaaa cgcaagagtc
     5521 ttctctgtct cgacaagccc agtttctatt ggtctcctta aacctgtctt gtaaccttga
     5581 tacttacctg atccaacggc tgcgacttgg ggcagggggt cctaggggtg gtctggccag
     5641 gagccgcggg ggcggactgc gcggtgccca ggagcacagc tgcgctctca ggcacgacac
     5701 gactcctccg ctgcccaccg cagactgagg cagcgctgag tcgccggcgc cgcagcgcag
     5761 atggtcgcgc ccgtgccccc ctatctcgcg cctcgcgtgg tgcggtccgg ctgggccggc
     5821 ggcggcgcgg acgcgaccaa ggtggccggg aaggggagtt tgcgggggac cggcgagtga
     5881 cgtcagcgcg ccttcagtgc tgaggcggcg gtggcgcgcg ccgccaggcg ggggcgaagg
     5941 cactgtccgc ggtgctgaag ctggcagtgc gcacgcgcct cgccgcatcc tgtttcccct
     6001 ccccctctct gataggggat gcgcaatttg gggaatgggg gttgggtgct tgtccagtgg
     6061 gtcggggtcg gtcgtcaatt taaatactga aaagggcgaa ttccagcaca ctggagctcg
     6121 gatcttccta gagcatggct acgtagataa gtagcatggc gggttaatca ttaactacaa
     6181 ggaaccccta gtgatggagt tggccactcc ctctctgcgc gctcgctcgc tcactgaggc
     6241 cgggcgacca aaggtcgccc gacgcccggg ctttgcccgg gcggcctcag tgagcgagcg
     6301 agcgcgcagc ctaattaagg ccttaattaa atctggcgta atcatggtca tagctgtttc
     6361 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     6421 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     6481 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     6541 ggagaggcgg tttgcgtatt ggg
//
