LOCUS       sequence-287199-        6628 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  piggyBac vector for expressing a Turquoise calcium sensor (GECI) 
            that reports with lifetime and intensity changes.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6628)
  AUTHORS   van der Linden FH, Mahlandt EK, Arts JJG, Beumer J, Puschhof J, de 
            Man SMA, Chertkova AO, Ponsioen B, Clevers H, van Buul JD, Postma M,
            Gadella TWJ Jr, Goedhart J
  TITLE     A turquoise fluorescence lifetime-based biosensor for quantitative 
            imaging of intracellular calcium.
  JOURNAL   Nat Commun. 2021 Dec 9;12(1):7159. doi: 10.1038/s41467-021-27249-w.
  PUBMED    34887382
REFERENCE   2  (bases 1 to 6628)
  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: "2021-12-9"; volume: "12(1):7159. doi"; pages: " 
            10.1038/s41467-021-27249-w"
COMMENT     Sequence Label: pPB-3xnls-Tq-Ca-FLITS
FEATURES             Location/Qualifiers
     source          1..6628
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   13..47
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        440..975
                     /label=mCMV promoter
                     /note="mouse cytomegalovirus (CMV) immediate early 
                     promoter"
     intron          999..1941
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1898..1918
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1996..2016
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2020..2040
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2044..2064
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      2077..2086
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2098..2154
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     CDS             2179..2427
                     /codon_start=1
                     /product="C-terminal fragment of mVenus for use in 
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VC155
                     /translation="DKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNH
                     YLSTQSKLSKDPNEKRDHMVLLEFVTAAGITLGMDELY"
     primer_bind     2367..2388
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2491..2512)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2752..2771)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(3393..3411)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3394..3411)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3431..3448)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3437..3661
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(3793..4381)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3873..3892)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4456..4590
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4493..4512)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4547..4566
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(4595..5194)
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(4678..4698)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     5175..5194
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     repeat_region   5597..5659
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     CDS             complement(join(5751..6542,6543..6611))
                     /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     6374..6393
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 ggcgcgcctt aaccctagaa agatagtctg cgtaaaattg acgcatgcat tcttgaaata
       61 ttgctctctc tttctaaata gcgcgaatcc gtcgctgtgc atttaggaca tctcagtcgc
      121 cgcttggagc tcccgtgagg cgtgcttgtc aatgcggtaa gtgtcactga ttttgaacta
      181 taacgaccgc gtgagtcaaa atgacgcatg attatctttt acgtgacttt taagatttaa
      241 ctcatacgat aattatattg ttatttcatg ttctacttac gtgataactt attatatata
      301 tattttcttg ttatagatat catcgataac aggaaagttc cattggagcc aagtacattg
      361 agtcaatagg gactttccaa tgggttttgc ccagtacata aggtcaatgg gaggtaagcc
      421 aatgggtttt tcccattact ggcacgtata ctgagtcatt agggactttc caatgggttt
      481 tgcccagtac ataaggtcaa taggggtgaa tcaacaggaa agttccattg gagccaagta
      541 cactgagtca atagggactt tccattgggt tttgcccagt acaaaaggtc aatagggggt
      601 gagtcaatgg gtttttccca ttattggcac gtacataagg tcaatagggg tgagtcattg
      661 ggtttttcca gccaatttaa ttaaaacgcc atgtactttc ccaccattga cgtcaatggg
      721 ctattgaaac taatgcaacg tgacctttaa acggtacttt cccatagctg attaatggga
      781 aagtaccgtt ctcgagccaa tacacgtcaa tgggaagtga aagggcagcc aaaacgtaac
      841 accgccccgg ttttcccctg gaaattccat attggcacgc attctattgg ctgagctgcg
      901 ttctacgtgg gtataagagg cgcgaccagc gtcggtaccg tcgcagtctt cggtctgacc
      961 accgtagaac gcagagctcc tcgctgcagg caagcttggt aagtgccgtg tgtggttccc
     1021 gcgggcctgg cctctttacg ggttatggcc cttgcgtgcc ttgaattact tccacgcccc
     1081 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
     1141 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
     1201 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
     1261 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
     1321 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc
     1381 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca
     1441 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg
     1501 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga
     1561 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc
     1621 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc
     1681 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg
     1741 agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc
     1801 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg
     1861 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa
     1921 agtttttttc ttccatttca gggatccact agtaacggcc gccagtgtgc tggaattccc
     1981 accatgggat cagatccaaa aaagaagaga aaggtagacc cgaagaaaaa gcgtaaggtc
     2041 gatcccaaaa agaaacggaa agtgggatca ccggtcgcca ccatggtcga ctcttcacgt
     2101 cgtaagtgga ataaggcagg tcacgcagtc agagctatag gtcggctgag ctcacccgtg
     2161 gttgtctata tcaccgccga caagcagaag aacggcatca aggccaactt caagatccgc
     2221 cacaacatcg aggacggcgg cgtgcagctc gccgaccact accagcagaa cacccccatc
     2281 ggcgacggcc ccgtgctgct gcccgacaac cactacctga gcacccagtc caagctgagc
     2341 aaagacccca acgagaagcg cgatcacatg gtcctgctgg agttcgtgac cgccgccggg
     2401 atcactctcg gcatggacga gctgtaccag ggtggcagcg gtggcatggt gagcaagggc
     2461 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     2521 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     2581 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     2641 tcctggggcg tgcagtgctt cgcccgctac cccgaccaca tgaagcagca cgacttcttc
     2701 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     2761 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     2821 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     2881 tactatagcg acaacacgcg tgaccaactg actgaagagc agatcgcaga atttaaagag
     2941 gctttctccc tatttgacaa ggacggggat gggacgataa caaccaagga gctggggacg
     3001 gtgatgcggt ctctggggca gaaccccaca gaagcagagc tgcaggacat gatcaatgaa
     3061 gtagatgccg acggtgacgg cacattcgac ttccctgagt tcctgacgat gatggcaaga
     3121 aaaatgaatg acacagacag tgaagaggaa attagagaag cgttccgcgt gtttgataag
     3181 gacggcaatg gctacatcgg cgcagcagag cttcgccacg tgatgacaga ccttggagag
     3241 aagttaacag atgaggaggt tgatgaaatg atcagggtag cagacatcga tggggatggt
     3301 caggtaaact acgaagagtt tgtccaaatg atgacagcga agtaggtgta caagtaaagc
     3361 ggccgctcga gcatgcatct agagggccct attctatagt gtcacctaaa tgctagagct
     3421 cgctgatcag cctcgactgt gccttctagt tgccagccat ctgttgtttg cccctccccc
     3481 gtgccttcct tgaccctgga aggtgccact cccactgtcc tttcctaata aaatgaggaa
     3541 attgcatcgc attgtctgag taggtgtcat tctattctgg ggggtggggt ggggcaggac
     3601 agcaaggggg aggattggga agacaatagc aggcatgctg gggatgcggt gggctctatg
     3661 gcttctgagg cggaaagaac cagtggcggt aatacggtta tccacagaat caggggataa
     3721 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3781 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     3841 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3901 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3961 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     4021 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     4081 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     4141 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     4201 gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct
     4261 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     4321 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     4381 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     4441 agggattttg gtcataactt gtttattgca gcttataatg gttacaaata aagcaatagc
     4501 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     4561 ctcatcaatg tatcttatca tgtctggatc cgcttcaggc accgggcttg cgggtcatgc
     4621 accaggtgcg cggtccttcg ggcacctcga cgtcggcggt gacggtgaag ccgagccgct
     4681 cgtagaaggg gaggttgcgg ggcgcggagg tctccaggaa ggcgggcacc ccggcgcgct
     4741 cggccgcctc cactccgggg agcacgacgg cgctgcccag acccttgccc tggtggtcgg
     4801 gcgagacgcc gacggtggcc aggaaccacg cgggctcctt gggccggtgc ggcgccagga
     4861 ggccttccat ctgttgctgc gcggccagcc tggaaccgct caactcggcc atgcgcgggc
     4921 cgatctcggc gaacaccgcc cccgcttcga cgctctccgg cgtggtccag accgccaccg
     4981 cggcgccgtc gtccgcgacc cacaccttgc cgatgtcgag cccgacgcgc gtgaggaaga
     5041 gttcttgcag ctcggtgacc cgctcgatgt ggcggtccgg gtcgacggtg tggcgcgtgg
     5101 cggggtagtc ggcgaacgcg gcggcgaggg tgcgtacggc ccgggggacg tcgtcgcggg
     5161 tggcgaggcg caccgtgggc ttgtactcgg tcatggtggc ctgcagagtc gctctgtgtt
     5221 cgaggccaca cgcgtcacct taatatgcga agtggacctg ggaccgcgcc gccccgactg
     5281 catctgcgtg ttttcgccaa tgacaagacg ctgggcgggg tttgtgtcat catagaacta
     5341 aagacatgca aatatatttc ttccggggac accgccagca aacgcgagca acgggccacg
     5401 gggatgaagc agctggctag ctaaaagttt tgttacttta tagaagaaat tttgagtttt
     5461 tgtttttttt taataaataa ataaacataa ataaattgtt tgttgaattt attattagta
     5521 tgtaagtgta aatataataa aacttaatat ctattcaaat taataaataa acctcgatat
     5581 acagaccgat aaaacacatg cgtcaatttt acgcatgatt atctttaacg tacgtcacaa
     5641 tatgattatc tttctagggt taattcgaac agctggttct ttccgcctca ggactcttcc
     5701 tttttcaata aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc
     5761 ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga
     5821 ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca
     5881 atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc
     5941 ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat
     6001 tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc
     6061 attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt
     6121 tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc
     6181 ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg
     6241 gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt
     6301 gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg
     6361 gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga
     6421 aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg
     6481 taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg
     6541 tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt
     6601 tgaatactca tactcttcct ttttcaat
//
