LOCUS       sequence-389371-        7361 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector for a low affinity calcium indicator 
            exposed to the cell surface.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7361)
  AUTHORS   Valiente-Gabioud AA, Garteizgogeascoa Suner I, Idziak A, Fabritius 
            A, Basquin J, Angibaud J, Nagerl UV, Singh SP, Griesbeck O
  TITLE     Fluorescent sensors for imaging of interstitial calcium.
  JOURNAL   Nat Commun. 2023 Oct 5;14(1):6220. doi: 10.1038/s41467-023-41928-w.
  PUBMED    37798285
REFERENCE   2  (bases 1 to 7361)
  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: "Nat 
            Commun."; date: "2023-10-5"; volume: "14(1):6220. doi"; pages: " 
            10.1038/s41467-023-41928-w"
COMMENT     Sequence Label: GreenT-EC.c - Display - CyRFP1
FEATURES             Location/Qualifiers
     source          1..7361
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(96..115)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        287..666
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        667..870
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     821..841
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     915..934
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        915..933
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     sig_peptide     959..1018
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             1031..1054
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1890..1899
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2066..2212
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     CDS             2255..2956
                     /codon_start=1
                     /product="monomeric cyan-excitable red fluorescent protein 
                     (Laviv et al., 2016)"
                     /label=mCyRFP1
                     /translation="MVSKGEELIKENMRSKLYLEGSVNGHQFKCTHEGEGKPYEGKQTA
                     RIKVVEGGPLPFAFDILATMFMYGSKVFIKYPADLPDYFKQSFPEGFTWERVMVFEDGG
                     VLTATQDTSLQDGELIYNVKLRGVNFPANGPVMQKKTLGWEPSTETMYPADGGLEGRCD
                     KKLKLVGGGHLHVNFKTTYKSKKPVKMPGVHYVDRRLERIKEADNETYVEQYEHAVARY
                     SNLGGGMDELYK"
     primer_bind     complement(3004..3021)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3010..3234
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3280..3708
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3367..3386)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3577..3598
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3717..3737)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3722..4051
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3902..4037
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3964..3983
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4118..4912
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4172..4191)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4782..4801
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5086..5207
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5123..5142)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5177..5196
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5256..5272)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5256..5272)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5269..5291)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5280..5296
                     /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(join(5304..5310,5311..5328,5329..5334))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5349..5370
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5487..5504)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5658..6246)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5738..5757)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6417..7208,7209..7277))
                     /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     7040..7059
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(7278..7361,1..21))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc
       61 gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag
      121 ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa
      181 atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt
      241 aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg
      301 actagttatt aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc
      361 cgcgttacat aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca
      421 ttgacgtcaa taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt
      481 caatgggtgg agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg
      541 ccaagtacgc cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag
      601 tacatgacct tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt
      661 accatggtga tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg
      721 ggatttccaa gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa
      781 cgggactttc caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt
      841 gtacggtggg aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac
      901 tggcttatcg aaattaatac gactcactat agggagaccc aagctggcta gcaccgccat
      961 ggagacagac acactcctgc tatgggtact gctgctctgg gttccaggtt ccactggtga
     1021 cggatccggt gattacaagg acgacgatga caagggtggt accggtggct ctggtagcag
     1081 tggcggtagc atggtctcca agggggagga ggacaacatg gggagcctgc cagctaccca
     1141 cgagctgcat atctttggat ctattaacgg ggtggatttc gacatggttg gacaaggtag
     1201 cgggaatcct aatgatgggt atgaagagct taaccttaaa agcactaagg gcgatctgca
     1261 attttcacct tggattctcg tcccccacat tgggtatggg ttccatcaat atctccctta
     1321 cccggatgga atgtctccct tccaagcggc tatggtagat ggtagtggtt accaagttca
     1381 caggactatg caattcgaag acggcgcctc attgactgtg aactaccgat acacttacga
     1441 aggatctcac ataaaaggtg aagcgcaggt gaagggcacg ggatttccag cagatgggcc
     1501 ggtaatgact aactcactta ctgcggcaga cttggggtgg gattcagaag aggaactttc
     1561 cgaatgtttt agaatttttg attttgacgg taacggattc attgaccgcg aagagttcgg
     1621 tgacataatc agattgactg gggagcaact tacggatgag gacgtagacg aaatttttgg
     1681 cgactcagat acggataaga attcaggccg gatcgacttt gatgaatttc tgaaaatggt
     1741 cgaaaatgtt cagctcacgg acaaccgaac aacgaaaaag acctatccaa atgacaagac
     1801 aattatctca actttgaaat ggagctacac cacggacaac ggcaagaggt atcggtccac
     1861 cgctatcact atttatacat ttgctaaacc catggcggct aactacctca agaatcaacc
     1921 tatgtacgtg tttcgcaaga ctgagcttaa acatagcaaa accgaattga atttcaagga
     1981 gtggcagaag gcgtttacgg acgtaatggg gatggatgag ctttataagg gtggtaccgg
     2041 tggctctggt agcagtggcg gtagcgctgt gggccaggac acgcaggagg tcatcgtggt
     2101 gccacactcc ttgcccttta aggtggtggt gatctcagcc atcctggccc tggtggtgct
     2161 caccatcatc tcccttatca tcctcatcat gctttggcag aagaagccac gtggtggtac
     2221 tggcggtagc ggttctagtg gcggttctga tatcatggtg agcaagggcg aggagctgat
     2281 caaggagaac atgagaagca agctgtacct ggaaggcagc gtgaacggcc accagttcaa
     2341 gtgcacccac gaaggggagg gcaagcccta cgagggcaag cagaccgcca ggatcaaggt
     2401 ggtggaggga ggccccctgc cgttcgcatt cgacatcctg gccaccatgt ttatgtacgg
     2461 gagcaaggtg ttcatcaagt accccgccga cctccccgat tattttaagc agtccttccc
     2521 tgagggcttc acatgggaga gagtcatggt gttcgaagac gggggcgtgc tgaccgccac
     2581 ccaggacacc agcctccagg acggcgagct catctacaac gtcaagctca gaggggtgaa
     2641 cttcccagcc aacggccccg tgatgcagaa gaaaacactg ggctgggagc ccagcaccga
     2701 gaccatgtac cccgctgacg gcggcctgga aggcagatgc gacaagaagc tgaagctcgt
     2761 gggcgggggc cacctgcacg tcaacttcaa gaccacatac aagtccaaga aacccgtgaa
     2821 gatgcccggc gtccactacg tggaccgcag actggaaaga atcaaggagg ccgacaacga
     2881 gacctacgtc gagcagtacg agcacgctgt ggccagatac tccaacctgg gcggaggcat
     2941 ggacgagctg tacaagtaag cggccgctcg agtctagagg gcccgtttaa acccgctgat
     3001 cagcctcgac tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt
     3061 ccttgaccct ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat
     3121 cgcattgtct gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg
     3181 gggaggattg ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg
     3241 aggcggaaag aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat
     3301 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     3361 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     3421 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     3481 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     3541 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     3601 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     3661 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa
     3721 tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag
     3781 catgcatctc aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag
     3841 aagtatgcaa agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc
     3901 catcccgccc ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt
     3961 ttttatttat gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg
     4021 aggctttttt ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt
     4081 cggatctgat caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca
     4141 cgcaggttct ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac
     4201 aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt
     4261 tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc
     4321 gtggctggcc acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg
     4381 aagggactgg ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc
     4441 tcctgccgag aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc
     4501 ggctacctgc ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat
     4561 ggaagccggt cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc
     4621 cgaactgttc gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca
     4681 tggcgatgcc tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga
     4741 ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat
     4801 tgctgaagag cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc
     4861 tcccgattcg cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact
     4921 ctggggttcg aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc
     4981 accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg
     5041 atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca
     5101 gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt
     5161 tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata
     5221 ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     5281 tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg
     5341 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     5401 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     5461 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     5521 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     5581 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     5641 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     5701 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     5761 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     5821 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     5881 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     5941 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     6001 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     6061 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     6121 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     6181 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     6241 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     6301 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     6361 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     6421 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     6481 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     6541 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     6601 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     6661 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     6721 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     6781 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     6841 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     6901 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     6961 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     7021 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     7081 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     7141 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     7201 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     7261 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     7321 tgtctcatga gcggatacat atttgaatgt atttagaaaa a
//
