LOCUS       sequence-423234-        6669 bp    DNA     circular SYN 15-APR-2025
DEFINITION  barcoded assay, cAMP/Ca2+ sensor.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6669)
  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 6669)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 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_CRE-CMV-BC0490-luc2
FEATURES             Location/Qualifiers
     source          1..6669
                     /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    complement(334..354)
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        370..407
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     404..428
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    490..510
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     promoter        537..555
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     promoter        complement(619..637)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(661..685)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      743..752
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             749..2401
                     /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             2405..2452
                     /codon_start=1
                     /product="non-ORF yeast peptide conferring 
                     ubiquitin-dependent degradation"
                     /label=hCL1
                     /note="human codon-optimized"
                     /translation="ACKNWFSSLSHFVIHL"
     CDS             2456..2575
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     polyA_signal    2624..2745
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2635..2654)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2689..2708
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        2939..3296
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2939..3130
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2962..2982)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3147..3282
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3209..3228
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3327..4364
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             4343..4351
                     /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    4388..4436
                     /note="synthetic polyadenylation signal"
     primer_bind     complement(4592..4609)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4763..5351)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4843..4862)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5551..6342,6343..6411))
                     /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    6516..6564
                     /note="synthetic polyadenylation signal"
     misc_feature    6578..6669
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     6618..6637
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggcctaactg gccggtacct gagctcgcta gcctcgagga tatcaaacaa gtttgtacaa
       61 aaaagcaggc tccctaactg gccggtaccg agctcttacg cgtgctagcg caccagacag
      121 tgacgtcagc tgccagatcc catggccgtc atactgtgac gtctttcaga caccccattg
      181 acgtcaatgg gagaacagat ctgccgcccc gactagcgca ccagacagtg acgtcagctg
      241 ccagatccca tggccgtcat actgtgacgt ctttcagaca ccccattgac gtcaatggga
      301 gaacagatct gccgccccga ctagcggggg gctcaacttt tctatacaaa gttgcggtac
      361 ccgggtcgag taggcgtgta cggtgggagg cctatataag cagagctcgt ttagtgaacc
      421 gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga caccgggacc
      481 gatccagccc aactttgtat aataaagttg agctagttgc taagtctgcc gagtagaatt
      541 aaccctcact aaagggtagg tgacactatt tacacattta cacattctat ctggctgttc
      601 tatactcaaa cttttctacc tatagtgagt cgtattagta gacgcgagtc aatgcatgta
      661 cgacagcttt cttgtacaaa gtggttcgat atcaagatct ggcctcggcg gccaagcttg
      721 gcaatccggt actgttggta aagccaccat ggaagatgcc aaaaacatta agaagggccc
      781 agcgccattc tacccactcg aagacgggac cgccggcgag cagctgcaca aagccatgaa
      841 gcgctacgcc ctggtgcccg gcaccatcgc ctttaccgac gcacatatcg aggtggacat
      901 tacctacgcc gagtacttcg agatgagcgt tcggctggca gaagctatga agcgctatgg
      961 gctgaataca aaccatcgga tcgtggtgtg cagcgagaat agcttgcagt tcttcatgcc
     1021 cgtgttgggt gccctgttca tcggtgtggc tgtggcccca gctaacgaca tctacaacga
     1081 gcgcgagctg ctgaacagca tgggcatcag ccagcccacc gtcgtattcg tgagcaagaa
     1141 agggctgcaa aagatcctca acgtgcaaaa gaagctaccg atcatacaaa agatcatcat
     1201 catggatagc aagaccgact accagggctt ccaaagcatg tacaccttcg tgacttccca
     1261 tttgccaccc ggcttcaacg agtacgactt cgtgcccgag agcttcgacc gggacaaaac
     1321 catcgccctg atcatgaaca gtagtggcag taccggattg cccaagggcg tagccctacc
     1381 gcaccgcacc gcttgtgtcc gattcagtca tgcccgcgac cccatcttcg gcaaccagat
     1441 catccccgac accgctatcc tcagcgtggt gccatttcac cacggcttcg gcatgttcac
     1501 cacgctgggc tacttgatct gcggctttcg ggtcgtgctc atgtaccgct tcgaggagga
     1561 gctattcttg cgcagcttgc aagactataa gattcaatct gccctgctgg tgcccacact
     1621 atttagcttc ttcgctaaga gcactctcat cgacaagtac gacctaagca acttgcacga
     1681 gatcgccagc ggcggggcgc cgctcagcaa ggaggtaggt gaggccgtgg ccaaacgctt
     1741 ccacctacca ggcatccgcc agggctacgg cctgacagaa acaaccagcg ccattctgat
     1801 cacccccgaa ggggacgaca agcctggcgc agtaggcaag gtggtgccct tcttcgaggc
     1861 taaggtggtg gacttggaca ccggtaagac actgggtgtg aaccagcgcg gcgagctgtg
     1921 cgtccgtggc cccatgatca tgagcggcta cgttaacaac cccgaggcta caaacgctct
     1981 catcgacaag gacggctggc tgcacagcgg cgacatcgcc tactgggacg aggacgagca
     2041 cttcttcatc gtggaccggc tgaagagcct gatcaaatac aagggctacc aggtagcccc
     2101 agccgaactg gagagcatcc tgctgcaaca ccccaacatc ttcgacgccg gggtcgccgg
     2161 cctgcccgac gacgatgccg gcgagctgcc cgccgcagtc gtcgtgctgg aacacggtaa
     2221 aaccatgacc gagaaggaga tcgtggacta tgtggccagc caggttacaa ccgccaagaa
     2281 gctgcgcggt ggtgttgtgt tcgtggacga ggtgcctaaa ggactgaccg gcaagttgga
     2341 cgcccgcaag atccgcgaga ttctcattaa ggccaagaag ggcggcaaga tcgccgtgta
     2401 atctgcttgc aagaactggt tcagtagctt aagccacttt gtgatccacc ttaacagcca
     2461 cggcttccct cccgaggtgg aggagcaggc cgccggcacc ctgcccatga gctgcgccca
     2521 ggagagcggc atggatagac accctgctgc ttgcgccagc gccaggatca acgtctaact
     2581 gcagtctaga gtcggggcgg ccggccgctt cgagcagaca tgataagata cattgatgag
     2641 tttggacaaa ccacaactag aatgcagtga aaaaaatgct ttatttgtga aatttgtgat
     2701 gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa caacaattgc
     2761 attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag caagtaaaac
     2821 ctctacaaat gtggtaaaat cgataaggat ccgtttgcgt attgggcgct cttccgctga
     2881 tctgcgcagc accatggcct gaaataacct ctgaaagagg aacttggtta gctaccttct
     2941 gaggcggaaa gaaccagctg tggaatgtgt gtcagttagg gtgtggaaag tccccaggct
     3001 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc aggtgtggaa
     3061 agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa
     3121 ccatagtccc gcccctaact ccgcccatcc cgcccctaac tccgcccagt tccgcccatt
     3181 ctccgcccca tggctgacta atttttttta tttatgcaga ggccgaggcc gcctctgcct
     3241 ctgagctatt ccagaagtag tgaggaggct tttttggagg cctaggcttt tgcaaaaagc
     3301 tcgattcttc tgacactagc gccaccatga agaagcccga actcaccgct accagcgttg
     3361 aaaaatttct catcgagaag ttcgacagtg tgagcgacct gatgcagttg tcggagggcg
     3421 aagagagccg agccttcagc ttcgatgtcg gcggacgcgg ctatgtactg cgggtgaata
     3481 gctgcgctga tggcttctac aaagaccgct acgtgtaccg ccacttcgcc agcgctgcac
     3541 tacccatccc cgaagtgttg gacatcggcg agttcagcga gagcctgaca tactgcatca
     3601 gtagacgcgc ccaaggcgtt actctccaag acctccccga aacagagctg cctgctgtgt
     3661 tacagcctgt cgccgaagct atggatgcta ttgccgccgc cgacctcagt caaaccagcg
     3721 gcttcggccc attcgggccc caaggcatcg gccagtacac aacctggcgg gatttcattt
     3781 gcgccattgc tgatccccat gtctaccact ggcagaccgt gatggacgac accgtgtccg
     3841 ccagcgtagc tcaagccctg gacgaactga tgctgtgggc cgaagactgt cccgaggtgc
     3901 gccacctcgt ccatgccgac ttcggcagca acaacgtcct gaccgacaac ggccgcatca
     3961 ccgccgtaat cgactggtcc gaagctatgt tcggggacag tcagtacgag gtggccaaca
     4021 tcttcttctg gcggccctgg ctggcttgca tggagcagca gactcgctac ttcgagcgcc
     4081 ggcatcccga gctggccggc agccctcgtc tgcgagccta catgctgcgc atcggcctgg
     4141 atcagctcta ccagagcctc gtggacggca acttcgacga tgctgcctgg gctcaaggcc
     4201 gctgcgatgc catcgtccgc agcggggccg gcaccgtcgg tcgcacacaa atcgctcgcc
     4261 ggagcgcagc cgtatggacc gacggctgcg tcgaggtgct ggccgacagc ggcaaccgcc
     4321 ggcccagtac acgaccgcgc gctaaggagg taggtcgagt ttaaactcta gaaccggtca
     4381 tggccgcaat aaaatatctt tattttcatt acatctgtgt gttggttttt tgtgtgttcg
     4441 aactagatgc tgtcgaccga tgcccttgag agccttcaac ccagtcagct ccttccggtg
     4501 ggcgcggggc atgactatcg tcgccgcact tatgactgtc ttctttatca tgcaactcgt
     4561 aggacaggtg ccggcagcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4621 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     4681 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     4741 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     4801 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     4861 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     4921 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     4981 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5041 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5101 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5161 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5221 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5281 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5341 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5401 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5461 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5521 gcggccgcaa atgctaaacc actgcagtgg ttaccagtgc ttgatcagtg aggcaccgat
     5581 ctcagcgatc tgcctatttc gttcgtccat agtggcctga ctccccgtcg tgtagatcac
     5641 tacgattcgt gagggcttac catcaggccc cagcgcagca atgatgccgc gagagccgcg
     5701 ttcaccggcc cccgatttgt cagcaatgaa ccagccagca gggagggccg agcgaagaag
     5761 tggtcctgct actttgtccg cctccatcca gtctatgagc tgctgtcgtg atgctagagt
     5821 aagaagttcg ccagtgagta gtttccgaag agttgtggcc attgctactg gcatcgtggt
     5881 atcacgctcg tcgttcggta tggcttcgtt caactctggt tcccagcggt caagccgggt
     5941 cacatgatca cccatattat gaagaaatgc agtcagctcc ttagggcctc cgatcgttgt
     6001 cagaagtaag ttggccgcgg tgttgtcgct catggtaatg gcagcactac acaattctct
     6061 taccgtcatg ccatccgtaa gatgcttttc cgtgaccggc gagtactcaa ccaagtcgtt
     6121 ttgtgagtag tgtatacggc gaccaagctg ctcttgcccg gcgtctatac gggacaacac
     6181 cgcgccacat agcagtactt tgaaagtgct catcatcggg aatcgttctt cggggcggaa
     6241 agactcaagg atcttgccgc tattgagatc cagttcgata tagcccactc ttgcacccag
     6301 ttgatcttca gcatctttta ctttcaccag cgtttcgggg tgtgcaaaaa caggcaagca
     6361 aaatgccgca aagaagggaa tgagtgcgac acgaaaatgt tggatgctca tactcgtcct
     6421 ttttcaatat tattgaagca tttatcaggg ttactagtac gtctctcaag gataagtaag
     6481 taatattaag gtacgggagg tattggacag gccgcaataa aatatcttta ttttcattac
     6541 atctgtgtgt tggttttttg tgtgaatcga tagtactaac atacgctctc catcaaaaca
     6601 aaacgaaaca aaacaaacta gcaaaatagg ctgtccccag tgcaagtgca ggtgccagaa
     6661 catttctct
//
