LOCUS       sequence-382346-        6575 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 6575)
  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 6575)
  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 - GPI
FEATURES             Location/Qualifiers
     source          1..6575
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(86..105)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        277..656
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        657..860
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     811..831
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     905..924
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        905..923
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1030..1053
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      1889..1898
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     primer_bind     complement(2208..2225)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2214..2438
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2484..2912
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2571..2590)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2781..2802
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2921..2941)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        2926..3255
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3106..3241
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3168..3187
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3322..4116
                     /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(3376..3395)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3986..4005
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4290..4411
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4327..4346)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4381..4400
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4460..4476)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4460..4476)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4473..4495)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4484..4500
                     /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(4508..4514,4515..4532,4533..4538))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4553..4574
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4691..4708)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4862..5450)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4942..4961)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5621..6412,6413..6481))
                     /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     6244..6263
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6482..6575,1..11))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tcgacggatc gggagatctc
       61 ccgatcccct atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt
      121 atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg cgcgagcaaa atttaagcta
      181 caacaaggca aggcttgacc gacaattgca tgaagaatct gcttagggtt aggcgttttg
      241 cgctgcttcg cgatgtacgg gccagatata cgcgttgaca ttgattattg actagttatt
      301 aatagtaatc aattacgggg tcattagttc atagcccata tatggagttc cgcgttacat
      361 aacttacggt aaatggcccg cctggctgac cgcccaacga cccccgccca ttgacgtcaa
      421 taatgacgta tgttcccata gtaacgccaa tagggacttt ccattgacgt caatgggtgg
      481 agtatttacg gtaaactgcc cacttggcag tacatcaagt gtatcatatg ccaagtacgc
      541 cccctattga cgtcaatgac ggtaaatggc ccgcctggca ttatgcccag tacatgacct
      601 tatgggactt tcctacttgg cagtacatct acgtattagt catcgctatt accatggtga
      661 tgcggttttg gcagtacatc aatgggcgtg gatagcggtt tgactcacgg ggatttccaa
      721 gtctccaccc cattgacgtc aatgggagtt tgttttggca ccaaaatcaa cgggactttc
      781 caaaatgtcg taacaactcc gccccattga cgcaaatggg cggtaggcgt gtacggtggg
      841 aggtctatat aagcagagct ctctggctaa ctagagaacc cactgcttac tggcttatcg
      901 aaattaatac gactcactat agggagaccc aagctggcta gcaccgccat ggtagagatg
      961 ctgccaactg tcgctgtgct ggtcttggca gtgtccgtgg ttgccaagga taacaccacg
     1021 ggatccggtg attacaagga cgacgatgac aagggtggta ccggtggctc tggtagcagt
     1081 ggcggtagca tggtctccaa gggggaggag gacaacatgg ggagcctgcc agctacccac
     1141 gagctgcata tctttggatc tattaacggg gtggatttcg acatggttgg acaaggtagc
     1201 gggaatccta atgatgggta tgaagagctt aaccttaaaa gcactaaggg cgatctgcaa
     1261 ttttcacctt ggattctcgt cccccacatt gggtatgggt tccatcaata tctcccttac
     1321 ccggatggaa tgtctccctt ccaagcggct atggtagatg gtagtggtta ccaagttcac
     1381 aggactatgc aattcgaaga cggcgcctca ttgactgtga actaccgata cacttacgaa
     1441 ggatctcaca taaaaggtga agcgcaggtg aagggcacgg gatttccagc agatgggccg
     1501 gtaatgacta actcacttac tgcggcagac ttggggtggg attcagaaga ggaactttcc
     1561 gaatgtttta gaatttttga ttttgacggt aacgacggat tcattgaccg cgaagagttc
     1621 ggtgacataa tcagattgac tggggagcaa cttacggatg aggacgtaga cgaaattttt
     1681 ggcgactcag atacggataa gaatggccgg atcgactttg atgaatttct gaaaatggtc
     1741 gaaaatgttc agctcacgga caaccgaaca acgaaaaaga cctatccaaa tgacaagaca
     1801 attatctcaa ctttgaaatg gagctacacc acggacaacg gcaagaggta tcggtccacc
     1861 gctatcacta cttatacatt tgctaaaccc atggcggcta actacctcaa gaatcaacct
     1921 atgtacgtgt ttcgcaagac tgagcttaaa catagcaaaa ccgaattgaa tttcaaggag
     1981 tggcagaagg cgtttacgga cgtaatgggg atggatgagc tttataaggg tggtaccggt
     2041 ggctctggta gcagtggcgg tagcaatggc ggcataagcc tgctggttca gaacacatcc
     2101 tggatgctgc tgctgctgct ttccctctcc ctcctccaag ccctggactt catttctctg
     2161 taagcggccg ctcgagtcta gagggcccgt ttaaacccgc tgatcagcct cgactgtgcc
     2221 ttctagttgc cagccatctg ttgtttgccc ctcccccgtg ccttccttga ccctggaagg
     2281 tgccactccc actgtccttt cctaataaaa tgaggaaatt gcatcgcatt gtctgagtag
     2341 gtgtcattct attctggggg gtggggtggg gcaggacagc aagggggagg attgggaaga
     2401 caatagcagg catgctgggg atgcggtggg ctctatggct tctgaggcgg aaagaaccag
     2461 ctggggctct agggggtatc cccacgcgcc ctgtagcggc gcattaagcg cggcgggtgt
     2521 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg ctcctttcgc
     2581 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg
     2641 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgatta
     2701 gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc ctttgacgtt
     2761 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaaccctat
     2821 ctcggtctat tcttttgatt tataagggat tttgccgatt tcggcctatt ggttaaaaaa
     2881 tgagctgatt taacaaaaat ttaacgcgaa ttaattctgt ggaatgtgtg tcagttaggg
     2941 tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca tctcaattag
     3001 tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat gcaaagcatg
     3061 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     3121 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     3181 gccgaggccg cctctgcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     3241 ctaggctttt gcaaaaagct cccgggagct tgtatatcca ttttcggatc tgatcaagag
     3301 acaggatgag gatcgtttcg catgattgaa caagatggat tgcacgcagg ttctccggcc
     3361 gcttgggtgg agaggctatt cggctatgac tgggcacaac agacaatcgg ctgctctgat
     3421 gccgccgtgt tccggctgtc agcgcagggg cgcccggttc tttttgtcaa gaccgacctg
     3481 tccggtgccc tgaatgaact gcaggacgag gcagcgcggc tatcgtggct ggccacgacg
     3541 ggcgttcctt gcgcagctgt gctcgacgtt gtcactgaag cgggaaggga ctggctgcta
     3601 ttgggcgaag tgccggggca ggatctcctg tcatctcacc ttgctcctgc cgagaaagta
     3661 tccatcatgg ctgatgcaat gcggcggctg catacgcttg atccggctac ctgcccattc
     3721 gaccaccaag cgaaacatcg catcgagcga gcacgtactc ggatggaagc cggtcttgtc
     3781 gatcaggatg atctggacga agagcatcag gggctcgcgc cagccgaact gttcgccagg
     3841 ctcaaggcgc gcatgcccga cggcgaggat ctcgtcgtga cccatggcga tgcctgcttg
     3901 ccgaatatca tggtggaaaa tggccgcttt tctggattca tcgactgtgg ccggctgggt
     3961 gtggcggacc gctatcagga catagcgttg gctacccgtg atattgctga agagcttggc
     4021 ggcgaatggg ctgaccgctt cctcgtgctt tacggtatcg ccgctcccga ttcgcagcgc
     4081 atcgccttct atcgccttct tgacgagttc ttctgagcgg gactctgggg ttcgaaatga
     4141 ccgaccaagc gacgcccaac ctgccatcac gagatttcga ttccaccgcc gccttctatg
     4201 aaaggttggg cttcggaatc gttttccggg acgccggctg gatgatcctc cagcgcgggg
     4261 atctcatgct ggagttcttc gcccacccca acttgtttat tgcagcttat aatggttaca
     4321 aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt
     4381 gtggtttgtc caaactcatc aatgtatctt atcatgtctg tataccgtcg acctctagct
     4441 agagcttggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4501 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4561 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4621 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4681 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4741 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4801 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4861 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4921 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4981 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     5041 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     5101 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     5161 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     5221 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     5281 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     5341 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5401 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5461 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5521 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5581 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5641 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5701 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5761 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5821 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5881 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5941 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     6001 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     6061 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     6121 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     6181 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     6241 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     6301 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6361 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6421 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6481 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6541 acatatttga atgtatttag aaaaataaac aaata
//
