LOCUS       sequence-315521-        6425 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Signaling reporter island (SiRI) construct for spatially multiplexed
            imaging. Contains GFP-based fluorescent reporter GCaMP6f (calcium 
            indicator), Xpress tag, and S1 protein scaffold..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6425)
  AUTHORS   Linghu C, Johnson SL, Valdes PA, Shemesh OA, Park WM, Park D, 
            Piatkevich KD, Wassie AT, Liu Y, An B, Barnes SA, Celiker OT, Yao 
            CC, Yu CJ, Wang R, Adamala KP, Bear MF, Keating AE, Boyden ES
  TITLE     Spatial Multiplexing of Fluorescent Reporters for Imaging Signaling 
            Network Dynamics.
  JOURNAL   Cell. 2020 Nov 17. pii: S0092-8674(20)31399-4. doi: 
            10.1016/j.cell.2020.10.035.
  PUBMED    33232692
REFERENCE   2  (bases 1 to 6425)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Cell. 2020 
            Nov 17. pii: S0092-8674(20)31399-4. doi: 
            10.1016/j.cell.2020.10.035."
COMMENT     Sequence Label: pAAV-UBC-S1-GCaMP6f
FEATURES             Location/Qualifiers
     source          1..6425
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..399
                     /label=UbC promoter
                     /note="human ubiquitin C promoter"
     regulatory      408..417
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(651..659,660..677,678..680,681..713,714..716,717..740,
                     741..767,768..824,825..830,831..1100,1101..1118,1119..1553,
                     1554..1559,1560..2000)
                     /codon_start=1
                     /product="improved fluorescent protein-based calcium sensor
                     (Chen et al., 2013)"
                     /label=GCaMP6f
                     /translation="MGSHHHHHHGMASMTGGQQMGRDLYDDDDKDLATMVDSSRRKWNK
                     TGHAVRAIGRLSSLENVYIKADKQKNGIKANFKIRHNIEDGGVQLAYHYQQNTPIGDGP
                     VLLPDNHYLSVQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGTGGSMVSKGEE
                     LFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLT
                     YGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFKDDGNYKTRAEVKFEGDTLVNRIE
                     LKGIDFKEDGNILGHKLEYNLPDQLTEEQIAEFKEEFSLFDKDGDGTITTKELGTVMRS
                     LGQNPTEAELQDMINEVDADGDGTIDFPEFLTMMARKMKYRDTEEEIREAFGVFDKDGN
                     GYISAAELRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAK"
     regulatory      747..756
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     1037..1058
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1164..1185)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1425..1444)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     CDS             2219..2233
                     /codon_start=1
                     /product="tag for protein cleavage using Ni2+ ion (Dang et 
                     al., 2019)"
                     /label=SNAC tag
                     /translation="GSHHW"
     misc_feature    2667..3255
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2720..2740)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(3138..3149)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     polyA_signal    3298..3419
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3309..3328)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3363..3382
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   3581..3710
                     /label=AAV2 ITR
     rep_origin      complement(3947..4535)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4027..4046)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4706..5497,5498..5566))
                     /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     5329..5348
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5567..5671)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5697..6152)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5834..5855)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6046..6065
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     repeat_region   6224..6353
                     /label=AAV2 ITR
ORIGIN
        1 ggcctccgcg ccgggttttg gcgcctcccg cgggcgcccc cctcctcacg gcgagcgctg
       61 ccacgtcaga cgaagggcgc agcgagcgtc ctgatccttc cgcccggacg ctcaggacag
      121 cggcccgctg ctcataagac tcggccttag aaccccagta tcagcagaag gacattttag
      181 gacgggactt gggtgactct agggcactgg ttttctttcc agagagcgga acaggcgagg
      241 aaaagtagtc ccttctcggc gattctgcgg agggatctcc gtggggcggt gaacgccgat
      301 gattatataa ggacgcgccg ggtgtggcac agctagttcc gtcgcagccg ggatttgggt
      361 cgcggttctt gtttgtggat cgctgtgatc gtcacttggc gactagtgcc accatggcag
      421 aagcagaaag cgcactggag tatgcacaac aagcactgga gaaggctcag ctggcactcc
      481 aagctgcaag acaggctctc aaggctggag gatccggtgg aaccggtgga agtggaggga
      541 caggaggttc aggtggtact ggtggctctg gaggaacagg aggctccgga ggtacaggag
      601 ggtcaggagg gactggaggc tctggaggta ctggcggttc aggaggtacc atgggttctc
      661 atcatcatca tcatcatggt atggcttcca tgactggtgg acagcaaatg ggtcgggatc
      721 tgtacgacga tgacgataag gatctcgcca ccatggtcga ctcatcacgt cgtaagtgga
      781 ataagacagg tcacgcagtc agagctatag gtcggctgag ttcactcgag aacgtctata
      841 tcaaggccga caagcagaag aacggcatca aggcgaactt caagatccgc cacaacatcg
      901 aggacggcgg cgtgcagctc gcctaccact accagcagaa cacccccatc ggcgacggcc
      961 ccgtgctgct gcccgacaac cactacctga gcgtgcagtc caaactttcg aaagacccca
     1021 acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg atcactctcg
     1081 gcatggacga gctgtacaag ggcggaaccg gagggagcat ggtgagcaag ggcgaggagc
     1141 tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac ggccacaagt
     1201 tcagcgtgtc cggcgagggt gagggcgatg ccacctacgg caagctgacc ctgaagttca
     1261 tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc ctgacctacg
     1321 gcgtgcagtg cttcagccgc taccccgacc acatgaagca gcacgacttc ttcaagtccg
     1381 ccatgcccga aggctacatc caggagcgca ccatcttctt caaggacgac ggcaactaca
     1441 agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc gagctgaagg
     1501 gcatcgactt caaggaggac ggcaacatcc tggggcacaa gctggagtac aacctgccgg
     1561 accaactgac tgaagagcag atcgcagaat ttaaagagga gttctcccta tttgacaagg
     1621 acggggatgg gacaataaca accaaggagc tggggacggt gatgcggtct ctggggcaga
     1681 accccacaga agcagagctg caggacatga tcaatgaagt agatgccgac ggtgacggca
     1741 caatcgactt ccctgagttc ctgacaatga tggcaagaaa aatgaaatac agggacacgg
     1801 aagaagaaat tagagaagcg ttcggtgtgt ttgataagga tggcaatggc tacatcagtg
     1861 cagcagagct tcgccacgtg atgacaaacc ttggagagaa gttaacagat gaagaggttg
     1921 atgaaatgat cagggaagca gacatcgatg gggatggtca ggtaaactac gaagagtttg
     1981 tacaaatgat gacagcgaag ggaggttcag gtggaaccgg tggaagtgga ggtaccatga
     2041 agatggagga gctgttcaag aagcataaga ttgtggctgt gctgagagct aactctgtgg
     2101 aggaagctaa gaagaaggct ctggctgtgt tcctgggagg cgttcacctg atcgagatta
     2161 ctttcacagt cccagatgcc gataccgtga tcaaggaact gtctttcctg aaagaaatgg
     2221 gagccatcat tggagctggc accgtcacct ccgtcgaaca atgtaggaaa gcagttgagt
     2281 ccggtgctga gttcatcgtc tctccacacc tggacgagga gatttctcag ttctgcaaag
     2341 agaaaggtgt gttctacatg cctggcgtca tgacacctac tgaactcgtc aaagccatga
     2401 agctcggaca taccatcctg aagctgttcc caggagaagt ggtgggacca cagttcgtga
     2461 aagccatgaa aggacccttc cctaatgtga agtttgtgcc cactggtggt gtgaatctgg
     2521 ataacgtgtg tgagtggttc aaggcaggcg ttctcgctgt cggagttggt tctgcactcg
     2581 tgaagggtac acctgtggaa gtcgctgaga aggcaaaggc tttcgttgag aagattagag
     2641 gatgtactga gtaaaagctt atcgataatc aacctctgga ttacaaaatt tgtgaaagat
     2701 tgactggtat tcttaactat gttgctcctt ttacgctatg tggatacgct gctttaatgc
     2761 ctttgtatca tgctattgct tcccgtatgg ctttcatttt ctcctccttg tataaatcct
     2821 ggttgctgtc tctttatgag gagttgtggc ccgttgtcag gcaacgtggc gtggtgtgca
     2881 ctgtgtttgc tgacgcaacc cccactggtt ggggcattgc caccacctgt cagctccttt
     2941 ccgggacttt cgctttcccc ctccctattg ccacggcgga actcatcgcc gcctgccttg
     3001 cccgctgctg gacaggggct cggctgttgg gcactgacaa ttccgtggtg ttgtcgggga
     3061 aatcatcgtc ctttccttgg ctgctcgcct gtgttgccac ctggattctg cgcgggacgt
     3121 ccttctgcta cgtcccttcg gccctcaatc cagcggacct tccttcccgc ggcctgctgc
     3181 cggctctgcg gcctcttccg cgtcttcgcc ttcgccctca gacgagtcgg atctcccttt
     3241 gggccgcctc cccgcatcga taccgtcgac ccgggcggcc gcttcgagca gacatgataa
     3301 gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt
     3361 gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta
     3421 acaacaacaa ttgcattcat tttatgtttc aggttcaggg ggagatgtgg gaggtttttt
     3481 aaagcaagta aaacctctac aaatgtggta aaatcgataa ggatcttcct agagcatggc
     3541 tacgtagata agtagcatgg cgggttaatc attaactaca aggaacccct agtgatggag
     3601 ttggccactc cctctctgcg cgctcgctcg ctcactgagg ccgggcgacc aaaggtcgcc
     3661 cgacgcccgg gctttgcccg ggcggcctca gtgagcgagc gagcgcgcag ctgcattaat
     3721 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     3781 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     3841 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     3901 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     3961 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4021 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4081 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4141 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     4201 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     4261 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     4321 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     4381 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     4441 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     4501 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     4561 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     4621 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     4681 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     4741 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     4801 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     4861 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     4921 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     4981 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5041 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5101 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     5161 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     5221 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     5281 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     5341 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     5401 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     5461 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     5521 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     5581 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     5641 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctaaat
     5701 tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca gctcattttt
     5761 taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga ccgagatagg
     5821 gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg actccaacgt
     5881 caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat caccctaatc
     5941 aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag ggagcccccg
     6001 atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga agaaagcgaa
     6061 aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa ccaccacacc
     6121 cgccgcgctt aatgcgccgc tacagggcgc gtcccattcg ccattcaggc tgcgcaactg
     6181 ttgggaaggg cgatcggtgc gggcctcttc gctattacgc cagctgcgcg ctcgctcgct
     6241 cactgaggcc gcccgggcaa agcccgggcg tcgggcgacc tttggtcgcc cggcctcagt
     6301 gagcgagcga gcgcgcagag agggagtggc caactccatc actaggggtt ccttgtagtt
     6361 aatgattaac ccgccatgct acttatctac gtagccatgc tctaggaaga tcgtaggtta
     6421 attaa
//
