LOCUS       sequence-308813-        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"
     promoter        185..720
                     /label=mCMV promoter
                     /note="mouse cytomegalovirus (CMV) immediate early 
                     promoter"
     intron          744..1686
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1643..1663
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1741..1761
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1765..1785
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1789..1809
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     regulatory      1822..1831
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1843..1899
                     /codon_start=1
                     /product="calmodulin-binding peptide from avian smooth 
                     muscle myosin light chain kinase"
                     /label=calmodulin-binding peptide
                     /translation="RRKWNKAGHAVRAIGRLSS"
     CDS             1924..2172
                     /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     complement(2236..2257)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2497..2516)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     promoter        complement(3138..3156)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3139..3156)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3176..3193)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3182..3406
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(3538..4126)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3618..3637)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     polyA_signal    4201..4335
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4238..4257)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4292..4311
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(4340..4939)
                     /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(4423..4443)
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     4920..4939
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     repeat_region   5342..5404
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     CDS             complement(join(5496..6287,6288..6356))
                     /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     6119..6138
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     repeat_region   6386..6420
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
ORIGIN
        1 tattgttatt tcatgttcta cttacgtgat aacttattat atatatattt tcttgttata
       61 gatatcatcg ataacaggaa agttccattg gagccaagta cattgagtca atagggactt
      121 tccaatgggt tttgcccagt acataaggtc aatgggaggt aagccaatgg gtttttccca
      181 ttactggcac gtatactgag tcattaggga ctttccaatg ggttttgccc agtacataag
      241 gtcaataggg gtgaatcaac aggaaagttc cattggagcc aagtacactg agtcaatagg
      301 gactttccat tgggttttgc ccagtacaaa aggtcaatag ggggtgagtc aatgggtttt
      361 tcccattatt ggcacgtaca taaggtcaat aggggtgagt cattgggttt ttccagccaa
      421 tttaattaaa acgccatgta ctttcccacc attgacgtca atgggctatt gaaactaatg
      481 caacgtgacc tttaaacggt actttcccat agctgattaa tgggaaagta ccgttctcga
      541 gccaatacac gtcaatggga agtgaaaggg cagccaaaac gtaacaccgc cccggttttc
      601 ccctggaaat tccatattgg cacgcattct attggctgag ctgcgttcta cgtgggtata
      661 agaggcgcga ccagcgtcgg taccgtcgca gtcttcggtc tgaccaccgt agaacgcaga
      721 gctcctcgct gcaggcaagc ttggtaagtg ccgtgtgtgg ttcccgcggg cctggcctct
      781 ttacgggtta tggcccttgc gtgccttgaa ttacttccac gcccctggct gcagtacgtg
      841 attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc ttgcgcttaa
      901 ggagcccctt cgcctcgtgc ttgagttgag gcctggcctg ggcgctgggg ccgccgcgtg
      961 cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct agccatttaa
     1021 aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt aaatgcgggc
     1081 caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg ggcccgtgcg
     1141 tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag aatcggacgg
     1201 gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc gtgtatcgcc
     1261 ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga aagatggccg
     1321 cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg agagcgggcg
     1381 ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc ttcatgtgac
     1441 tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt ttggagtacg
     1501 tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac tgagtgggtg
     1561 gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt tgcccttttt
     1621 gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt ttttcttcca
     1681 tttcagggat ccactagtaa cggccgccag tgtgctggaa ttcccaccat gggatcagat
     1741 ccaaaaaaga agagaaaggt agacccgaag aaaaagcgta aggtcgatcc caaaaagaaa
     1801 cggaaagtgg gatcaccggt cgccaccatg gtcgactctt cacgtcgtaa gtggaataag
     1861 gcaggtcacg cagtcagagc tataggtcgg ctgagctcac ccgtggttgt ctatatcacc
     1921 gccgacaagc agaagaacgg catcaaggcc aacttcaaga tccgccacaa catcgaggac
     1981 ggcggcgtgc agctcgccga ccactaccag cagaacaccc ccatcggcga cggccccgtg
     2041 ctgctgcccg acaaccacta cctgagcacc cagtccaagc tgagcaaaga ccccaacgag
     2101 aagcgcgatc acatggtcct actggagttc gtgaccgccg ccgggatcac tctcggcatg
     2161 gacgagctgt accagggtgg cagcggtggc atggtgagca agggcgagga gctgttcacc
     2221 ggggtggtgc ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg
     2281 tccggcgagg gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc
     2341 accggcaagc tgcccgtgcc ctggcccacc ctcgtgacca ccctgtcctg gggcgtgcag
     2401 tgcttcgccc gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc
     2461 gaaggctacg tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc
     2521 gccgaggtga agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac
     2581 ttcaaggagg acggcaacat cctggggcac aagctggagt acaactacta tagcgacaac
     2641 acgcgtgacc aactgactga agagcagatc gcagaattta aagaggcttt ctccctattt
     2701 gacaaggacg gggatgggac gataacaacc aaggagctgg ggacggtgat gcggtctctg
     2761 gggcagaacc ccacagaagc agagctgcag gacatgatca atgaagtaga tgccgacggt
     2821 gacggcacat tcgacttccc tgagttcctg acgatgatgg caagaaaaat gaatgacaca
     2881 gacagtgaag aggaaattag agaagcgttc cgcgtgtttg ataaggacgg caatggctac
     2941 atcggcgcag cagagcttcg ccacgtgatg acagaccttg gagagaagtt aacagatgag
     3001 gaggttgatg aaatgatcag ggtagcagac atcgatgggg atggtcaggt aaactacgaa
     3061 gagtttgtcc aaatgatgac agcgaagtag gtgtacaagt aaagcggccg ctcgagcatg
     3121 catctagagg gccctattct atagtgtcac ctaaatgcta gagctcgctg atcagcctcg
     3181 actgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
     3241 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt
     3301 ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
     3361 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa
     3421 agaaccagtg gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     3481 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     3541 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3601 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3661 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3721 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3781 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3841 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3901 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3961 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     4021 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     4081 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     4141 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     4201 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
     4261 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
     4321 tatcatgtct ggatccgctt caggcaccgg gcttgcgggt catgcaccag gtgcgcggtc
     4381 cttcgggcac ctcgacgtcg gcggtgacgg tgaagccgag ccgctcgtag aaggggaggt
     4441 tgcggggcgc ggaggtctcc aggaaggcgg gcaccccggc gcgctcggcc gcctccactc
     4501 cggggagcac gacggcgctg cccagaccct tgccctggtg gtcgggcgag acgccgacgg
     4561 tggccaggaa ccacgcgggc tccttgggcc ggtgcggcgc caggaggcct tccatctgtt
     4621 gctgcgcggc cagcctggaa ccgctcaact cggccatgcg cgggccgatc tcggcgaaca
     4681 ccgcccccgc ttcgacgctc tccggcgtgg tccagaccgc caccgcggcg ccgtcgtccg
     4741 cgacccacac cttgccgatg tcgagcccga cgcgcgtgag gaagagttct tgcagctcgg
     4801 tgacccgctc gatgtggcgg tccgggtcga cggtgtggcg cgtggcgggg tagtcggcga
     4861 acgcggcggc gagggtgcgt acggcccggg ggacgtcgtc gcgggtggcg aggcgcaccg
     4921 tgggcttgta ctcggtcatg gtggcctgca gagtcgctct gtgttcgagg ccacacgcgt
     4981 caccttaata tgcgaagtgg acctgggacc gcgccgcccc gactgcatct gcgtgttttc
     5041 gccaatgaca agacgctggg cggggtttgt gtcatcatag aactaaagac atgcaaatat
     5101 atttcttccg gggacaccgc cagcaaacgc gagcaacggg ccacggggat gaagcagctg
     5161 gctagctaaa agttttgtta ctttatagaa gaaattttga gtttttgttt ttttttaata
     5221 aataaataaa cataaataaa ttgtttgttg aatttattat tagtatgtaa gtgtaaatat
     5281 aataaaactt aatatctatt caaattaata aataaacctc gatatacaga ccgataaaac
     5341 acatgcgtca attttacgca tgattatctt taacgtacgt cacaatatga ttatctttct
     5401 agggttaatt cgaacagctg gttctttccg cctcaggact cttccttttt caataaatca
     5461 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     5521 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     5581 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     5641 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     5701 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     5761 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc
     5821 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     5881 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     5941 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     6001 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     6061 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat
     6121 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     6181 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     6241 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     6301 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     6361 ttcctttttc aatggcgcgc cttaacccta gaaagatagt ctgcgtaaaa ttgacgcatg
     6421 cattcttgaa atattgctct ctctttctaa atagcgcgaa tccgtcgctg tgcatttagg
     6481 acatctcagt cgccgcttgg agctcccgtg aggcgtgctt gtcaatgcgg taagtgtcac
     6541 tgattttgaa ctataacgac cgcgtgagtc aaaatgacgc atgattatct tttacgtgac
     6601 ttttaagatt taactcatac gataatta
//
