LOCUS       sequence-423225-        6673 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  barcoded assay, Ca2+ sensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6673)
  AUTHORS   Popovic L, Rossner MJ, Wehr MC
  TITLE     Protocol for identifying properties of ERBB receptor antagonists 
            using the barcoded ERBBprofiler assay.
  JOURNAL   STAR Protoc. 2024 Apr 16;5(2):102987. doi: 
            10.1016/j.xpro.2024.102987.
  PUBMED    38635397
REFERENCE   2  (bases 1 to 6673)
  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: "STAR 
            Protoc."; date: "2024-04-16"; volume: "5(2):102987. doi"; pages: " 
            10.1016/j.xpro.2024.102987"
COMMENT     Sequence Label: pGL4_NFAT-RE-CMV-BC0530-luc2
FEATURES             Location/Qualifiers
     source          1..6673
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    47..71
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    120..149
                     /label=NFAT binding site
                     /bound_moiety="NFAT transcription factor"
                     /note="NFAT binding site from the human interleukin 2 
                     (IL-2) gene (Mattila et al., 1990)"
     protein_bind    150..179
                     /label=NFAT binding site
                     /bound_moiety="NFAT transcription factor"
                     /note="NFAT binding site from the human interleukin 2 
                     (IL-2) gene (Mattila et al., 1990)"
     protein_bind    180..209
                     /label=NFAT binding site
                     /bound_moiety="NFAT transcription factor"
                     /note="NFAT binding site from the human interleukin 2 
                     (IL-2) gene (Mattila et al., 1990)"
     protein_bind    233..262
                     /label=NFAT binding site
                     /bound_moiety="NFAT transcription factor"
                     /note="NFAT binding site from the human interleukin 2 
                     (IL-2) gene (Mattila et al., 1990)"
     protein_bind    263..292
                     /label=NFAT binding site
                     /bound_moiety="NFAT transcription factor"
                     /note="NFAT binding site from the human interleukin 2 
                     (IL-2) gene (Mattila et al., 1990)"
     protein_bind    293..322
                     /label=NFAT binding site
                     /bound_moiety="NFAT transcription factor"
                     /note="NFAT binding site from the human interleukin 2 
                     (IL-2) gene (Mattila et al., 1990)"
     protein_bind    complement(338..358)
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        374..411
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     408..432
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    494..514
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        541..559
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(622..641)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(623..641)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(665..689)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      747..756
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             753..2405
                     /codon_start=1
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="synthetic luc2 version of the luciferase gene"
                     /translation="MEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDA
                     HIEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAP
                     ANDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQS
                     MYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHA
                     RDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYK
                     IQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGY
                     GLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMS
                     GYVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESIL
                     LQHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVV
                     FVDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     CDS             2409..2456
                     /codon_start=1
                     /product="non-ORF yeast peptide conferring 
                     ubiquitin-dependent degradation"
                     /label=hCL1
                     /note="human codon-optimized"
                     /translation="ACKNWFSSLSHFVIHL"
     CDS             2460..2579
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     polyA_signal    2628..2749
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2639..2658)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2693..2712
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        2943..3300
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2943..3134
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2966..2986)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3151..3286
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3213..3232
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3331..4368
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             4347..4355
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     polyA_signal    4392..4440
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4596..4613)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4767..5355)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4847..4866)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5555..6346,6347..6415))
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     polyA_signal    6520..6568
                     /note="synthetic polyadenylation signal"
     misc_feature    6582..6673
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     6622..6641
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggcctaactg gccggtacct gagctcgcta gcctcgagga tatcaaacaa gtttgtacaa
       61 aaaagcaggc tcccaagtgc aggtgccaga acatttctct ggcctaactg gccggtaccg
      121 gaggaaaaac tgtttcatac agaaggcgtg gaggaaaaac tgtttcatac agaaggcgtg
      181 gaggaaaaac tgtttcatac agaaggcgtg ctagtcctaa ctggccggta ccggaggaaa
      241 aactgtttca tacagaaggc gtggaggaaa aactgtttca tacagaaggc gtggaggaaa
      301 aactgtttca tacagaaggc gtgctagcgg ggggctacaa cttttctata caaagttgcg
      361 gtacccgggt cgagtaggcg tgtacggtgg gaggcctata taagcagagc tcgtttagtg
      421 aaccgtcaga tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg
      481 gaccgatcca gcccaacttt gtataataaa gttgagctag ttgctaagtc tgccgagtag
      541 aattaaccct cactaaaggg taggtgacac tatatcacaa atctaaatcc ttttctcgga
      601 ctctttacat tactaatcaa tccctatagt gagtcgtatt agtagacgcg agtcaatgca
      661 tgtacgacag ctttcttgta caaagtggtt cgatatcaag atctggcctc ggcggccaag
      721 cttggcaatc cggtactgtt ggtaaagcca ccatggaaga tgccaaaaac attaagaagg
      781 gcccagcgcc attctaccca ctcgaagacg ggaccgccgg cgagcagctg cacaaagcca
      841 tgaagcgcta cgccctggtg cccggcacca tcgcctttac cgacgcacat atcgaggtgg
      901 acattaccta cgccgagtac ttcgagatga gcgttcggct ggcagaagct atgaagcgct
      961 atgggctgaa tacaaaccat cggatcgtgg tgtgcagcga gaatagcttg cagttcttca
     1021 tgcccgtgtt gggtgccctg ttcatcggtg tggctgtggc cccagctaac gacatctaca
     1081 acgagcgcga gctgctgaac agcatgggca tcagccagcc caccgtcgta ttcgtgagca
     1141 agaaagggct gcaaaagatc ctcaacgtgc aaaagaagct accgatcata caaaagatca
     1201 tcatcatgga tagcaagacc gactaccagg gcttccaaag catgtacacc ttcgtgactt
     1261 cccatttgcc acccggcttc aacgagtacg acttcgtgcc cgagagcttc gaccgggaca
     1321 aaaccatcgc cctgatcatg aacagtagtg gcagtaccgg attgcccaag ggcgtagccc
     1381 taccgcaccg caccgcttgt gtccgattca gtcatgcccg cgaccccatc ttcggcaacc
     1441 agatcatccc cgacaccgct atcctcagcg tggtgccatt tcaccacggc ttcggcatgt
     1501 tcaccacgct gggctacttg atctgcggct ttcgggtcgt gctcatgtac cgcttcgagg
     1561 aggagctatt cttgcgcagc ttgcaagact ataagattca atctgccctg ctggtgccca
     1621 cactatttag cttcttcgct aagagcactc tcatcgacaa gtacgaccta agcaacttgc
     1681 acgagatcgc cagcggcggg gcgccgctca gcaaggaggt aggtgaggcc gtggccaaac
     1741 gcttccacct accaggcatc cgccagggct acggcctgac agaaacaacc agcgccattc
     1801 tgatcacccc cgaaggggac gacaagcctg gcgcagtagg caaggtggtg cccttcttcg
     1861 aggctaaggt ggtggacttg gacaccggta agacactggg tgtgaaccag cgcggcgagc
     1921 tgtgcgtccg tggccccatg atcatgagcg gctacgttaa caaccccgag gctacaaacg
     1981 ctctcatcga caaggacggc tggctgcaca gcggcgacat cgcctactgg gacgaggacg
     2041 agcacttctt catcgtggac cggctgaaga gcctgatcaa atacaagggc taccaggtag
     2101 ccccagccga actggagagc atcctgctgc aacaccccaa catcttcgac gccggggtcg
     2161 ccggcctgcc cgacgacgat gccggcgagc tgcccgccgc agtcgtcgtg ctggaacacg
     2221 gtaaaaccat gaccgagaag gagatcgtgg actatgtggc cagccaggtt acaaccgcca
     2281 agaagctgcg cggtggtgtt gtgttcgtgg acgaggtgcc taaaggactg accggcaagt
     2341 tggacgcccg caagatccgc gagattctca ttaaggccaa gaagggcggc aagatcgccg
     2401 tgtaatctgc ttgcaagaac tggttcagta gcttaagcca ctttgtgatc caccttaaca
     2461 gccacggctt ccctcccgag gtggaggagc aggccgccgg caccctgccc atgagctgcg
     2521 cccaggagag cggcatggat agacaccctg ctgcttgcgc cagcgccagg atcaacgtct
     2581 aactgcagtc tagagtcggg gcggccggcc gcttcgagca gacatgataa gatacattga
     2641 tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa tgctttattt gtgaaatttg
     2701 tgatgctatt gctttatttg taaccattat aagctgcaat aaacaagtta acaacaacaa
     2761 ttgcattcat tttatgtttc aggttcaggg ggaggtgtgg gaggtttttt aaagcaagta
     2821 aaacctctac aaatgtggta aaatcgataa ggatccgttt gcgtattggg cgctcttccg
     2881 ctgatctgcg cagcaccatg gcctgaaata acctctgaaa gaggaacttg gttagctacc
     2941 ttctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3001 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3061 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3121 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3181 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     3241 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3301 aagctcgatt cttctgacac tagcgccacc atgaagaagc ccgaactcac cgctaccagc
     3361 gttgaaaaat ttctcatcga gaagttcgac agtgtgagcg acctgatgca gttgtcggag
     3421 ggcgaagaga gccgagcctt cagcttcgat gtcggcggac gcggctatgt actgcgggtg
     3481 aatagctgcg ctgatggctt ctacaaagac cgctacgtgt accgccactt cgccagcgct
     3541 gcactaccca tccccgaagt gttggacatc ggcgagttca gcgagagcct gacatactgc
     3601 atcagtagac gcgcccaagg cgttactctc caagacctcc ccgaaacaga gctgcctgct
     3661 gtgttacagc ctgtcgccga agctatggat gctattgccg ccgccgacct cagtcaaacc
     3721 agcggcttcg gcccattcgg gccccaaggc atcggccagt acacaacctg gcgggatttc
     3781 atttgcgcca ttgctgatcc ccatgtctac cactggcaga ccgtgatgga cgacaccgtg
     3841 tccgccagcg tagctcaagc cctggacgaa ctgatgctgt gggccgaaga ctgtcccgag
     3901 gtgcgccacc tcgtccatgc cgacttcggc agcaacaacg tcctgaccga caacggccgc
     3961 atcaccgccg taatcgactg gtccgaagct atgttcgggg acagtcagta cgaggtggcc
     4021 aacatcttct tctggcggcc ctggctggct tgcatggagc agcagactcg ctacttcgag
     4081 cgccggcatc ccgagctggc cggcagccct cgtctgcgag cctacatgct gcgcatcggc
     4141 ctggatcagc tctaccagag cctcgtggac ggcaacttcg acgatgctgc ctgggctcaa
     4201 ggccgctgcg atgccatcgt ccgcagcggg gccggcaccg tcggtcgcac acaaatcgct
     4261 cgccggagcg cagccgtatg gaccgacggc tgcgtcgagg tgctggccga cagcggcaac
     4321 cgccggccca gtacacgacc gcgcgctaag gaggtaggtc gagtttaaac tctagaaccg
     4381 gtcatggccg caataaaata tctttatttt cattacatct gtgtgttggt tttttgtgtg
     4441 ttcgaactag atgctgtcga ccgatgccct tgagagcctt caacccagtc agctccttcc
     4501 ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac
     4561 tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4621 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4681 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4741 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4801 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4861 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     4921 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     4981 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5041 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5101 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5161 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5221 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5281 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5341 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5401 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5461 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5521 gacagcggcc gcaaatgcta aaccactgca gtggttacca gtgcttgatc agtgaggcac
     5581 cgatctcagc gatctgccta tttcgttcgt ccatagtggc ctgactcccc gtcgtgtaga
     5641 tcactacgat tcgtgagggc ttaccatcag gccccagcgc agcaatgatg ccgcgagagc
     5701 cgcgttcacc ggcccccgat ttgtcagcaa tgaaccagcc agcagggagg gccgagcgaa
     5761 gaagtggtcc tgctactttg tccgcctcca tccagtctat gagctgctgt cgtgatgcta
     5821 gagtaagaag ttcgccagtg agtagtttcc gaagagttgt ggccattgct actggcatcg
     5881 tggtatcacg ctcgtcgttc ggtatggctt cgttcaactc tggttcccag cggtcaagcc
     5941 gggtcacatg atcacccata ttatgaagaa atgcagtcag ctccttaggg cctccgatcg
     6001 ttgtcagaag taagttggcc gcggtgttgt cgctcatggt aatggcagca ctacacaatt
     6061 ctcttaccgt catgccatcc gtaagatgct tttccgtgac cggcgagtac tcaaccaagt
     6121 cgttttgtga gtagtgtata cggcgaccaa gctgctcttg cccggcgtct atacgggaca
     6181 acaccgcgcc acatagcagt actttgaaag tgctcatcat cgggaatcgt tcttcggggc
     6241 ggaaagactc aaggatcttg ccgctattga gatccagttc gatatagccc actcttgcac
     6301 ccagttgatc ttcagcatct tttactttca ccagcgtttc ggggtgtgca aaaacaggca
     6361 agcaaaatgc cgcaaagaag ggaatgagtg cgacacgaaa atgttggatg ctcatactcg
     6421 tcctttttca atattattga agcatttatc agggttacta gtacgtctct caaggataag
     6481 taagtaatat taaggtacgg gaggtattgg acaggccgca ataaaatatc tttattttca
     6541 ttacatctgt gtgttggttt tttgtgtgaa tcgatagtac taacatacgc tctccatcaa
     6601 aacaaaacga aacaaaacaa actagcaaaa taggctgtcc ccagtgcaag tgcaggtgcc
     6661 agaacatttc tct
//
