LOCUS      pGL4_CRE-CMVmin-luc2        6669 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Reporter plasmid for cAMP/Calcium assays; firefly luciferase 
            reporter gene driven by 6x clustered CRE binding sites linked to a 
            minimal CMV promoter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6669)
  AUTHORS   Wu Y, von Hauff IV, Jensen N, Rossner MJ, Wehr MC
  TITLE     Improved Split TEV GPCR beta-arrestin-2 Recruitment Assays via 
            Systematic Analysis of Signal Peptide and beta-arrestin Binding 
            Motif Variants.
  JOURNAL   Biosensors (Basel). 2022 Dec 29;13(1):48. doi: 10.3390/bios13010048.
  PUBMED    36671883
REFERENCE   2  (bases 1 to 6669)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            //gt12365.cn
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Biosensors 
            (Basel)."; date: "2022-12-29"; volume: "13(1):48. doi"; pages: " 
            10.3390/bios13010048"
COMMENT     Sequence Label: pGL4_CRE-CMVmin-luc2
FEATURES             Location/Qualifiers
     source          1..6669
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..48)
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     enhancer        127..318
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     275..295
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     polyA_signal    512..633
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(549..568)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     603..622
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     CDS             complement(682..801)
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     CDS             complement(805..852)
                     /codon_start=1
                     /product="non-ORF yeast peptide conferring 
                     ubiquitin-dependent degradation"
                     /label=hCL1
                     /note="human codon-optimized"
                     /translation="ACKNWFSSLSHFVIHL"
     CDS             complement(856..2508)
                     /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"
     regulatory      2505..2514
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     protein_bind    2572..2596
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     2620..2639
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        2620..2638
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     promoter        complement(2702..2720)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     protein_bind    complement(2747..2767)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(2829..2853)
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     promoter        complement(2850..2887)
                     /label=minimal CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     protein_bind    2903..2923
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     protein_bind    complement(3186..3210)
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    3257..3348
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     complement(3289..3308)
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
     polyA_signal    3362..3410
                     /note="synthetic polyadenylation signal"
     CDS             join(3515..3583,3584..4375)
                     /codon_start=1
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     rep_origin      4575..5163
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5064..5083
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5317..5334
                     /label=L4440
                     /note="L4440 vector, forward primer"
     polyA_signal    5490..5538
                     /note="synthetic polyadenylation signal"
     CDS             complement(5562..6599)
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     EVGRV"
     RBS             5575..5583
                     /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)"
     promoter        complement(join(6630..6669,1..318))
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      join(6644..6669,1..110)
                     /label=SV40 ori
                     /note="SV40 origin of replication"
ORIGIN
        1 ttctggaata gctcagaggc agaggcggcc tcggcctctg cataaataaa aaaaattagt
       61 cagccatggg gcggagaatg ggcggaactg ggcggagtta ggggcgggat gggcggagtt
      121 aggggcggga ctatggttgc tgactaattg agatgcatgc tttgcatact tctgcctgct
      181 ggggagcctg gggactttcc acacctggtt gctgactaat tgagatgcat gctttgcata
      241 cttctgcctg ctggggagcc tggggacttt ccacacccta actgacacac attccacagc
      301 tggttctttc cgcctcagaa ggtagctaac caagttcctc tttcagaggt tatttcaggc
      361 catggtgctg cgcagatcag cggaagagcg cccaatacgc aaacggatcc ttatcgattt
      421 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
      481 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
      541 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
      601 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tgctcgaagc ggccggccgc
      661 cccgactcta gactgcagtt agacgttgat cctggcgctg gcgcaagcag cagggtgtct
      721 atccatgccg ctctcctggg cgcagctcat gggcagggtg ccggcggcct gctcctccac
      781 ctcgggaggg aagccgtggc tgttaaggtg gatcacaaag tggcttaagc tactgaacca
      841 gttcttgcaa gcagattaca cggcgatctt gccgcccttc ttggccttaa tgagaatctc
      901 gcggatcttg cgggcgtcca acttgccggt cagtccttta ggcacctcgt ccacgaacac
      961 aacaccaccg cgcagcttct tggcggttgt aacctggctg gccacatagt ccacgatctc
     1021 cttctcggtc atggttttac cgtgttccag cacgacgact gcggcgggca gctcgccggc
     1081 atcgtcgtcg ggcaggccgg cgaccccggc gtcgaagatg ttggggtgtt gcagcaggat
     1141 gctctccagt tcggctgggg ctacctggta gcccttgtat ttgatcaggc tcttcagccg
     1201 gtccacgatg aagaagtgct cgtcctcgtc ccagtaggcg atgtcgccgc tgtgcagcca
     1261 gccgtccttg tcgatgagag cgtttgtagc ctcggggttg ttaacgtagc cgctcatgat
     1321 catggggcca cggacgcaca gctcgccgcg ctggttcaca cccagtgtct taccggtgtc
     1381 caagtccacc accttagcct cgaagaaggg caccaccttg cctactgcgc caggcttgtc
     1441 gtccccttcg ggggtgatca gaatggcgct ggttgtttct gtcaggccgt agccctggcg
     1501 gatgcctggt aggtggaagc gtttggccac ggcctcacct acctccttgc tgagcggcgc
     1561 cccgccgctg gcgatctcgt gcaagttgct taggtcgtac ttgtcgatga gagtgctctt
     1621 agcgaagaag ctaaatagtg tgggcaccag cagggcagat tgaatcttat agtcttgcaa
     1681 gctgcgcaag aatagctcct cctcgaagcg gtacatgagc acgacccgaa agccgcagat
     1741 caagtagccc agcgtggtga acatgccgaa gccgtggtga aatggcacca cgctgaggat
     1801 agcggtgtcg gggatgatct ggttgccgaa gatggggtcg cgggcatgac tgaatcggac
     1861 acaagcggtg cggtgcggta gggctacgcc cttgggcaat ccggtactgc cactactgtt
     1921 catgatcagg gcgatggttt tgtcccggtc gaagctctcg ggcacgaagt cgtactcgtt
     1981 gaagccgggt ggcaaatggg aagtcacgaa ggtgtacatg ctttggaagc cctggtagtc
     2041 ggtcttgcta tccatgatga tgatcttttg tatgatcggt agcttctttt gcacgttgag
     2101 gatcttttgc agccctttct tgctcacgaa tacgacggtg ggctggctga tgcccatgct
     2161 gttcagcagc tcgcgctcgt tgtagatgtc gttagctggg gccacagcca caccgatgaa
     2221 cagggcaccc aacacgggca tgaagaactg caagctattc tcgctgcaca ccacgatccg
     2281 atggtttgta ttcagcccat agcgcttcat agcttctgcc agccgaacgc tcatctcgaa
     2341 gtactcggcg taggtaatgt ccacctcgat atgtgcgtcg gtaaaggcga tggtgccggg
     2401 caccagggcg tagcgcttca tggctttgtg cagctgctcg ccggcggtcc cgtcttcgag
     2461 tgggtagaat ggcgctgggc ccttcttaat gtttttggca tcttccatgg tggctttacc
     2521 aacagtaccg gattgccaag cttggccgcc gaggccagat cttgatatcg aaccactttg
     2581 tacaagaaag ctgtcgtaca tgcattgact cgcgtctact aatacgactc actataggga
     2641 ttgtaagatt gatttagaag tcgctgttag atgattttga gtatgatata gtgtcaccta
     2701 ccctttagtg agggttaatt ctactcggca gacttagcaa ctagctcaac tttattatac
     2761 aaagttgggc tggatcggtc ccggtgtctt ctatggaggt caaaacagcg tggatggcgt
     2821 ctccaggcga tctgacggtt cactaaacga gctctgctta tataggcctc ccaccgtaca
     2881 cgcctactcg acccgggtac cgcaactttg tatagaaaag ttgagccccc cgctagtcgg
     2941 ggcggcagat ctgttctccc attgacgtca atggggtgtc tgaaagacgt cacagtatga
     3001 cggccatggg atctggcagc tgacgtcact gtctggtgcg ctagtcgggg cggcagatct
     3061 gttctcccat tgacgtcaat ggggtgtctg aaagacgtca cagtatgacg gccatgggat
     3121 ctggcagctg acgtcactgt ctggtgcgct agcacgcgta agagctcggt accggccagt
     3181 tagggagcct gcttttttgt acaaacttgt ttgatatcct cgaggctagc gagctcaggt
     3241 accggccagt taggccagag aaatgttctg gcacctgcac ttgcactggg gacagcctat
     3301 tttgctagtt tgttttgttt cgttttgttt tgatggagag cgtatgttag tactatcgat
     3361 tcacacaaaa aaccaacaca cagatgtaat gaaaataaag atattttatt gcggcctgtc
     3421 caatacctcc cgtaccttaa tattacttac ttatccttga gagacgtact agtaaccctg
     3481 ataaatgctt caataatatt gaaaaaggac gagtatgagc atccaacatt ttcgtgtcgc
     3541 actcattccc ttctttgcgg cattttgctt gcctgttttt gcacaccccg aaacgctggt
     3601 gaaagtaaaa gatgctgaag atcaactggg tgcaagagtg ggctatatcg aactggatct
     3661 caatagcggc aagatccttg agtctttccg ccccgaagaa cgattcccga tgatgagcac
     3721 tttcaaagta ctgctatgtg gcgcggtgtt gtcccgtata gacgccgggc aagagcagct
     3781 tggtcgccgt atacactact cacaaaacga cttggttgag tactcgccgg tcacggaaaa
     3841 gcatcttacg gatggcatga cggtaagaga attgtgtagt gctgccatta ccatgagcga
     3901 caacaccgcg gccaacttac ttctgacaac gatcggaggc cctaaggagc tgactgcatt
     3961 tcttcataat atgggtgatc atgtgacccg gcttgaccgc tgggaaccag agttgaacga
     4021 agccataccg aacgacgagc gtgataccac gatgccagta gcaatggcca caactcttcg
     4081 gaaactactc actggcgaac ttcttactct agcatcacga cagcagctca tagactggat
     4141 ggaggcggac aaagtagcag gaccacttct tcgctcggcc ctccctgctg gctggttcat
     4201 tgctgacaaa tcgggggccg gtgaacgcgg ctctcgcggc atcattgctg cgctggggcc
     4261 tgatggtaag ccctcacgaa tcgtagtgat ctacacgacg gggagtcagg ccactatgga
     4321 cgaacgaaat aggcagatcg ctgagatcgg tgcctcactg atcaagcact ggtaaccact
     4381 gcagtggttt agcatttgcg gccgctgtca gaccaagttt actcatatat actttagatt
     4441 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     4501 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     4561 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     4621 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     4681 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     4741 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     4801 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     4861 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     4921 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     4981 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5041 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5101 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5161 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5221 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     5281 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     5341 gaagagcgct gccggcacct gtcctacgag ttgcatgata aagaagacag tcataagtgc
     5401 ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg actgggttga aggctctcaa
     5461 gggcatcggt cgacagcatc tagttcgaac acacaaaaaa ccaacacaca gatgtaatga
     5521 aaataaagat attttattgc ggccatgacc ggttctagag tttaaactcg acctacctcc
     5581 ttagcgcgcg gtcgtgtact gggccggcgg ttgccgctgt cggccagcac ctcgacgcag
     5641 ccgtcggtcc atacggctgc gctccggcga gcgatttgtg tgcgaccgac ggtgccggcc
     5701 ccgctgcgga cgatggcatc gcagcggcct tgagcccagg cagcatcgtc gaagttgccg
     5761 tccacgaggc tctggtagag ctgatccagg ccgatgcgca gcatgtaggc tcgcagacga
     5821 gggctgccgg ccagctcggg atgccggcgc tcgaagtagc gagtctgctg ctccatgcaa
     5881 gccagccagg gccgccagaa gaagatgttg gccacctcgt actgactgtc cccgaacata
     5941 gcttcggacc agtcgattac ggcggtgatg cggccgttgt cggtcaggac gttgttgctg
     6001 ccgaagtcgg catggacgag gtggcgcacc tcgggacagt cttcggccca cagcatcagt
     6061 tcgtccaggg cttgagctac gctggcggac acggtgtcgt ccatcacggt ctgccagtgg
     6121 tagacatggg gatcagcaat ggcgcaaatg aaatcccgcc aggttgtgta ctggccgatg
     6181 ccttggggcc cgaatgggcc gaagccgctg gtttgactga ggtcggcggc ggcaatagca
     6241 tccatagctt cggcgacagg ctgtaacaca gcaggcagct ctgtttcggg gaggtcttgg
     6301 agagtaacgc cttgggcgcg tctactgatg cagtatgtca ggctctcgct gaactcgccg
     6361 atgtccaaca cttcggggat gggtagtgca gcgctggcga agtggcggta cacgtagcgg
     6421 tctttgtaga agccatcagc gcagctattc acccgcagta catagccgcg tccgccgaca
     6481 tcgaagctga aggctcggct ctcttcgccc tccgacaact gcatcaggtc gctcacactg
     6541 tcgaacttct cgatgagaaa tttttcaacg ctggtagcgg tgagttcggg cttcttcatg
     6601 gtggcgctag tgtcagaaga atcgagcttt ttgcaaaagc ctaggcctcc aaaaaagcct
     6661 cctcactac
//