LOCUS       sequence-357328-        5326 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses GCaMP6f on the cytosolic side of the ER (P-450site).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5326)
  AUTHORS   Aryal SP, Xia M, Adindu E, Davis C, Ortinski PI, Richards CI
  TITLE     ER-GCaMP6f: An Endoplasmic Reticulum-Targeted Genetic Probe to 
            Measure Calcium Activity in Astrocytic Processes.
  JOURNAL   Anal Chem. 2022 Jan 21. doi: 10.1021/acs.analchem.1c04321.
  PUBMED    35061939
REFERENCE   2  (bases 1 to 5326)
  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: "Anal Chem. 
            2022 Jan 21. doi: 10.1021/acs.analchem.1c04321."
COMMENT     Sequence Label: ER-GCaMP6f
FEATURES             Location/Qualifiers
     source          1..5326
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        112..415
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        416..619
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     570..590
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      783..792
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             804..860
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKTGHAVRAIGRLSS"
     primer_bind     1073..1094
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(1200..1221)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1461..1480)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     polyA_signal    2165..2286
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2202..2221)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2256..2275
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(2293..2748)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2430..2451)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2642..2661
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2775..2879
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2881..3238
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2881..3072
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2904..2924)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3089..3224
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3273..4067
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             3273..4067
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(3327..3346)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3937..3956
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4255..4274)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    4299..4346
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4675..5263
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5164..5183
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccatgca ttagttatta
       61 atagtaatca attacggggt cattagttca tagcccatat atggagttcc gcgttacata
      121 acttacggta aatggcccgc ctggctgacc gcccaacgac ccccgcccat tgacgtcaat
      181 aatgacgtat gttcccatag taacgccaat agggactttc cattgacgtc aatgggtgga
      241 gtatttacgg taaactgccc acttggcagt acatcaagtg tatcatatgc caagtacgcc
      301 ccctattgac gtcaatgacg gtaaatggcc cgcctggcat tatgcccagt acatgacctt
      361 atgggacttt cctacttggc agtacatcta cgtattagtc atcgctatta ccatggtgat
      421 gcggttttgg cagtacatca atgggcgtgg atagcggttt gactcacggg gatttccaag
      481 tctccacccc attgacgtca atgggagttt gttttggcac caaaatcaac gggactttcc
      541 aaaatgtcgt aacaactccg ccccattgac gcaaatgggc ggtaggcgtg tacggtggga
      601 ggtctatata agcagagctg gtttagtgaa ccgtcagatc cgctagcgct accggactca
      661 gatctcgagc caccatgcat ccggatatgg accctgtggt cgtgctgggg ctgtgcctga
      721 gctgtctgct gctgctgagc ctgtggaagc agtcctacgg aggcgggttg gatccaccgg
      781 tcgccaccat ggtcgactca tcacgtcgta agtggaataa gacaggtcac gcagtcagag
      841 ctataggtcg gctgagctca ctcgagaacg tctatatcaa ggccgacaag cagaagaacg
      901 gcatcaaggc gaacttcaag atccgccaca acatcgagga cggcggcgtg cagctcgcct
      961 accactacca gcagaacacc cccatcggcg acggccccgt gctgctgccc gacaaccact
     1021 acctgagcgt gcagtccaaa ctttcgaaag accccaacga gaagcgcgat cacatggtcc
     1081 tgctggagtt cgtgaccgcc gccgggatca ctctcggcat ggacgagctg tacaagggcg
     1141 gtaccggagg gagcatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     1201 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggtgagg
     1261 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     1321 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     1381 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacatccagg
     1441 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     1501 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     1561 acatcctggg gcacaagctg gagtacaacc tgccggacca actgactgaa gagcagatcg
     1621 cagaatttaa agaggaattc tccctatttg acaaggacgg ggatgggaca ataacaacca
     1681 aggagctggg gacggtgatg cggtctctgg ggcagaaccc cacagaagca gagctgcagg
     1741 acatgatcaa tgaagtagat gccgacggtg acggcacaat cgacttccct gagttcctga
     1801 caatgatggc aagaaaaatg aaatacaggg acacggaaga agaaattaga gaagcgttcg
     1861 gtgtgtttga taaggatggc aatggctaca tcagtgcagc agagcttcgc cacgtgatga
     1921 caaaccttgg agagaagtta acagatgaag aggttgatga aatgatcagg gaagcagaca
     1981 tcgatgggga tggtcaggta aactacgaag agtttgtaca aatgatgaca gcgaagcttt
     2041 aaagcggccg cgactctaga tcataatcag ccataccaca tttgtagagg ttttacttgc
     2101 tttaaaaaac ctcccacacc tccccctgaa cctgaaacat aaaatgaatg caattgttgt
     2161 tgttaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     2221 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     2281 atcttaaggc gtaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt
     2341 aaatcagctc attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag
     2401 aatagaccga gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga
     2461 acgtggactc caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg
     2521 aaccatcacc ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc
     2581 ctaaagggag cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg
     2641 aagggaagaa agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc
     2701 gcgtaaccac cacacccgcc gcgcttaatg cgccgctaca gggcgcgtca ggtggcactt
     2761 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     2821 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagtc
     2881 ctgaggcgga aagaaccagc tgtggaatgt gtgtcagtta gggtgtggaa agtccccagg
     2941 ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccaggtgtgg
     3001 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     3061 aaccatagtc ccgcccctaa ctccgcccat cccgccccta actccgccca gttccgccca
     3121 ttctccgccc catggctgac taattttttt tatttatgca gaggccgagg ccgcctcggc
     3181 ctctgagcta ttccagaagt agtgaggagg cttttttgga ggcctaggct tttgcaaaga
     3241 tcgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga ttgcacgcag
     3301 gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa cagacaatcg
     3361 gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt ctttttgtca
     3421 agaccgacct gtccggtgcc ctgaatgaac tgcaagacga ggcagcgcgg ctatcgtggc
     3481 tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa gcgggaaggg
     3541 actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac cttgctcctg
     3601 ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt gatccggcta
     3661 cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact cggatggaag
     3721 ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg ccagccgaac
     3781 tgttcgccag gctcaaggcg agcatgcccg acggcgagga tctcgtcgtg acccatggcg
     3841 atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc atcgactgtg
     3901 gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt gatattgctg
     3961 aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc gccgctcccg
     4021 attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg ggactctggg
     4081 gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg attccaccgc
     4141 cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct
     4201 ccagcgcggg gatctcatgc tggagttctt cgcccaccct agggggaggc taactgaaac
     4261 acggaaggag acaataccgg aaggaacccg cgctatgacg gcaataaaaa gacagaataa
     4321 aacgcacggt gttgggtcgt ttgttcataa acgcggggtt cggtcccagg gctggcactc
     4381 tgtcgatacc ccaccgagac cccattgggg ccaatacgcc cgcgtttctt ccttttcccc
     4441 accccacccc ccaagttcgg gtgaaggccc agggctcgca gccaacgtcg gggcggcagg
     4501 ccctgccata gcctcaggtt actcatatat actttagatt gatttaaaac ttcattttta
     4561 atttaaaagg atctaggtga agatcctttt tgataatctc atgaccaaaa tcccttaacg
     4621 tgagttttcg ttccactgag cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga
     4681 tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt
     4741 ggtttgtttg ccggatcaag agctaccaac tctttttccg aaggtaactg gcttcagcag
     4801 agcgcagata ccaaatactg ttcttctagt gtagccgtag ttaggccacc acttcaagaa
     4861 ctctgtagca ccgcctacat acctcgctct gctaatcctg ttaccagtgg ctgctgccag
     4921 tggcgataag tcgtgtctta ccgggttgga ctcaagacga tagttaccgg ataaggcgca
     4981 gcggtcgggc tgaacggggg gttcgtgcac acagcccagc ttggagcgaa cgacctacac
     5041 cgaactgaga tacctacagc gtgagctatg agaaagcgcc acgcttcccg aagggagaaa
     5101 ggcggacagg tatccggtaa gcggcagggt cggaacagga gagcgcacga gggagcttcc
     5161 agggggaaac gcctggtatc tttatagtcc tgtcgggttt cgccacctct gacttgagcg
     5221 tcgatttttg tgatgctcgt caggggggcg gagcctatgg aaaaacgcca gcaacgcggc
     5281 ctttttacgg ttcctggcct tttgctggcc ttttgctcac atgttc
//
