LOCUS       sequence-389372-        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.b - Display - CyRFP1
FEATURES             Location/Qualifiers
     source          1..7361
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(68..87)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        259..638
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        639..842
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     793..813
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     887..906
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        887..905
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     sig_peptide     931..990
                     /codon_start=1
                     /product="leader sequence from mouse immunoglobulin kappa 
                     light chain"
                     /label=Ig-kappa leader
                     /translation="METDTLLLWVLLLWVPGSTG"
     CDS             1003..1026
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1862..1871
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2038..2184
                     /codon_start=1
                     /product="transmembrane domain from platelet derived growth
                     factor receptor beta"
                     /label=PDGFR-beta TM domain
                     /translation="AVGQDTQEVIVVPHSLPFKVVVISAILALVVLTIISLIILIMLWQ
                     KKPR"
     CDS             2227..2928
                     /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(2976..2993)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2982..3206
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3252..3680
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3339..3358)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3549..3570
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3689..3709)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3694..4023
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3874..4009
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3936..3955
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4090..4884
                     /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(4144..4163)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4754..4773
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5058..5179
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5095..5114)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5149..5168
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5228..5244)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5228..5244)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5241..5263)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5252..5268
                     /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(5276..5282,5283..5300,5301..5306))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5321..5342
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5459..5476)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5630..6218)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5710..5729)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6389..7180,7181..7249))
                     /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     7012..7031
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7250..7354)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ccccgaaaag tgccacctga cgtcgacgga tcgggagatc tcccgatccc ctatggtgca
       61 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatctgctc cctgcttgtg
      121 tgttggaggt cgctgagtag tgcgcgagca aaatttaagc tacaacaagg caaggcttga
      181 ccgacaattg catgaagaat ctgcttaggg ttaggcgttt tgcgctgctt cgcgatgtac
      241 gggccagata tacgcgttga cattgattat tgactagtta ttaatagtaa tcaattacgg
      301 ggtcattagt tcatagccca tatatggagt tccgcgttac ataacttacg gtaaatggcc
      361 cgcctggctg accgcccaac gacccccgcc cattgacgtc aataatgacg tatgttccca
      421 tagtaacgcc aatagggact ttccattgac gtcaatgggt ggagtattta cggtaaactg
      481 cccacttggc agtacatcaa gtgtatcata tgccaagtac gccccctatt gacgtcaatg
      541 acggtaaatg gcccgcctgg cattatgccc agtacatgac cttatgggac tttcctactt
      601 ggcagtacat ctacgtatta gtcatcgcta ttaccatggt gatgcggttt tggcagtaca
      661 tcaatgggcg tggatagcgg tttgactcac ggggatttcc aagtctccac cccattgacg
      721 tcaatgggag tttgttttgg caccaaaatc aacgggactt tccaaaatgt cgtaacaact
      781 ccgccccatt gacgcaaatg ggcggtaggc gtgtacggtg ggaggtctat ataagcagag
      841 ctctctggct aactagagaa cccactgctt actggcttat cgaaattaat acgactcact
      901 atagggagac ccaagctggc tagcaccgcc atggagacag acacactcct gctatgggta
      961 ctgctgctct gggttccagg ttccactggt gacggatccg gtgattacaa ggacgacgat
     1021 gacaagggtg gtaccggtgg ctctggtagc agtggcggta gcatggtctc caagggggag
     1081 gaggacaaca tggggagcct gccagctacc cacgagctgc atatctttgg atctattaac
     1141 ggggtggatt tcgacatggt tggacaaggt agcgggaatc ctaatgatgg gtatgaagag
     1201 cttaacctta aaagcactaa gggcgatctg caattttcac cttggattct cgtcccccac
     1261 attgggtatg ggttccatca atatctccct tacccggatg gaatgtctcc cttccaagcg
     1321 gctatggtag atggtagtgg ttaccaagtt cacaggacta tgcaattcga agacggcgcc
     1381 tcattgactg tgaactaccg atacacttac gaaggatctc acataaaagg tgaagcgcag
     1441 gtgaagggca cgggatttcc agcagatggg ccggtaatga ctaactcact tactgcggca
     1501 gacttggggt gggattcaga agaggaactt tccgaatgtt ttagaatttt tgattttgac
     1561 ggtaacggat tcattgaccg cgaagagttc ggtgacataa tcagattgac tggggagcaa
     1621 cttacggatg aggacgtaga cgaaattttt ggcgactcag atacggataa gaattcaggc
     1681 cggatcgact ttgatgaatt tctgaaaatg gtcgaaaatg ttcagctcac ggacaaccga
     1741 acaacgaaaa agacctatcc aaatgacaag acaattatct caactttgaa atggagctac
     1801 accacggaca acggcaagag gtatcggtcc accgctatca ctacttatac atttgctaaa
     1861 cccatggcgg ctaactacct caagaatcaa cctatgtacg tgtttcgcaa gactgagctt
     1921 aaacatagca aaaccgaatt gaatttcaag gagtggcaga aggcgtttac ggacgtaatg
     1981 gggatggatg agctttataa gggtggtacc ggtggctctg gtagcagtgg cggtagcgct
     2041 gtgggccagg acacgcagga ggtcatcgtg gtgccacact ccttgccctt taaggtggtg
     2101 gtgatctcag ccatcctggc cctggtggtg ctcaccatca tctcccttat catcctcatc
     2161 atgctttggc agaagaagcc acgtggtggt actggcggta gcggttctag tggcggttct
     2221 gatatcatgg tgagcaaggg cgaggagctg atcaaggaga acatgagaag caagctgtac
     2281 ctggaaggca gcgtgaacgg ccaccagttc aagtgcaccc acgaagggga gggcaagccc
     2341 tacgagggca agcagaccgc caggatcaag gtggtggagg gaggccccct gccgttcgca
     2401 ttcgacatcc tggccaccat gtttatgtac gggagcaagg tgttcatcaa gtaccccgcc
     2461 gacctccccg attattttaa gcagtccttc cctgagggct tcacatggga gagagtcatg
     2521 gtgttcgaag acgggggcgt gctgaccgcc acccaggaca ccagcctcca ggacggcgag
     2581 ctcatctaca acgtcaagct cagaggggtg aacttcccag ccaacggccc cgtgatgcag
     2641 aagaaaacac tgggctggga gcccagcacc gagaccatgt accccgctga cggcggcctg
     2701 gaaggcagat gcgacaagaa gctgaagctc gtgggcgggg gccacctgca cgtcaacttc
     2761 aagaccacat acaagtccaa gaaacccgtg aagatgcccg gcgtccacta cgtggaccgc
     2821 agactggaaa gaatcaagga ggccgacaac gagacctacg tcgagcagta cgagcacgct
     2881 gtggccagat actccaacct gggcggaggc atggacgagc tgtacaagta agcggccgct
     2941 cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt ctagttgcca
     3001 gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg ccactcccac
     3061 tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt gtcattctat
     3121 tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca atagcaggca
     3181 tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct ggggctctag
     3241 ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg
     3301 cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt tcttcccttc
     3361 ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc tccctttagg
     3421 gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg gtgatggttc
     3481 acgtagtggg ccatcgccct gatagacggt ttttcgccct ttgacgttgg agtccacgtt
     3541 ctttaatagt ggactcttgt tccaaactgg aacaacactc aaccctatct cggtctattc
     3601 ttttgattta taagggattt tgccgatttc ggcctattgg ttaaaaaatg agctgattta
     3661 acaaaaattt aacgcgaatt aattctgtgg aatgtgtgtc agttagggtg tggaaagtcc
     3721 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccagg
     3781 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3841 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     3901 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     3961 tctgcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4021 aaaaagctcc cgggagcttg tatatccatt ttcggatctg atcaagagac aggatgagga
     4081 tcgtttcgca tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag
     4141 aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc
     4201 cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg
     4261 aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc
     4321 gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg
     4381 ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct
     4441 gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg
     4501 aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat
     4561 ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgcgc
     4621 atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg
     4681 gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc
     4741 tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct
     4801 gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat
     4861 cgccttcttg acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga
     4921 cgcccaacct gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct
     4981 tcggaatcgt tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg
     5041 agttcttcgc ccaccccaac ttgtttattg cagcttataa tggttacaaa taaagcaata
     5101 gcatcacaaa tttcacaaat aaagcatttt tttcactgca ttctagttgt ggtttgtcca
     5161 aactcatcaa tgtatcttat catgtctgta taccgtcgac ctctagctag agcttggcgt
     5221 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca
     5281 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat
     5341 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     5401 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct
     5461 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa
     5521 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa
     5581 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc
     5641 tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga
     5701 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
     5761 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt
     5821 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctgggct
     5881 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg
     5941 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta
     6001 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct
     6061 acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
     6121 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt
     6181 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg atcttttcta
     6241 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat
     6301 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa
     6361 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct
     6421 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
     6481 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct
     6541 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg
     6601 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa
     6661 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt
     6721 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta
     6781 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
     6841 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta
     6901 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct
     6961 gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg
     7021 cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg gggcgaaaac
     7081 tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact
     7141 gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
     7201 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt
     7261 ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac atatttgaat
     7321 gtatttagaa aaataaacaa ataggggttc cgcgcacatt t
//
