LOCUS       sequence-244622-        7712 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Circularly permutated firefly luciferase with PPVs cleavage site 
            (Luciferase reporter for detection of proteolytic activity).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7712)
  AUTHORS   Fink T, Lonzaric J, Praznik A, Plaper T, Merljak E, Leben K, Jerala 
            N, Lebar T, Strmsek Z, Lapenta F, Bencina M, Jerala R
  TITLE     Design of fast proteolysis-based signaling and logic circuits in 
            mammalian cells.
  JOURNAL   Nat Chem Biol. 2019 Feb;15(2):115-122. doi: 
            10.1038/s41589-018-0181-6. Epub 2018 Dec 10.
  PUBMED    30531965
REFERENCE   2  (bases 1 to 7712)
  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"; doi: 
            "10.1038/s41589-018-0181-6"; journalName: "Nat Chem Biol"; date: 
            "2019-02"; volume: "15"; issue: "2"; pages: "115-122"
COMMENT     Sequence Label: cycLuc_PPVs
FEATURES             Location/Qualifiers
     source          1..7712
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(44..63)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        615..818
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     769..789
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     863..882
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             922..939
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             3070..3117
                     /codon_start=1
                     /product="non-ORF yeast peptide conferring 
                     ubiquitin-dependent degradation"
                     /label=hCL1
                     /note="human codon-optimized"
                     /translation="ACKNWFSSLSHFVIHL"
     CDS             3121..3240
                     /codon_start=1
                     /product="PEST degradation sequence from mouse ornithine 
                     decarboxylase"
                     /label=hPEST
                     /note="human codon-optimized"
                     /translation="SHGFPPEVEEQAAGTLPMSCAQESGMDRHPAACASARINV"
     promoter        complement(3265..3283)
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     complement(3266..3283)
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     primer_bind     complement(3303..3320)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3309..3533
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3579..4007
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3666..3685)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3876..3897
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4016..4036)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4021..4350
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4201..4336
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4263..4282
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4417..5211
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4471..4490)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5081..5100
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5385..5506
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5422..5441)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5476..5495
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5555..5571)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5555..5571)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5568..5590)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5579..5595
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(5603..5609,5610..5627,5628..5633))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5648..5669
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5786..5803)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5957..6545)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6037..6056)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6716..7507,7508..7576))
                     /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     7339..7358
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7577..7681)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gcttggtacc
      901 gagctcgcta gcgccaccat gcaccaccac caccaccaca tgatcaaaat agccacacgt
      961 aaatatttag gcaaacaaaa tgtctatgac attggagttg agcgcgacca taattttgca
     1021 ctcaaaaatg gcttcatagc ttctaattgt ttcaatgata cttatagata tattgacacc
     1081 gctatcctca gcgtggtgcc atttcaccac ggcttcggca tgttcaccac gctgggctac
     1141 ttgatctgcg gctttcgggt cgtgctcatg taccgcttcg aggaggagct attcttgcgc
     1201 agcttgcaag actataagat tcaatctgcc ctgctggtgc ccacactatt tagcttcttc
     1261 gctaagagca ctctcatcga caagtacgac ctaagcaact tgcacgagat cgccagcggc
     1321 ggggcgccgc tcagcaagga ggtaggtgag gccgtggcca aacgcttcca cctaccaggc
     1381 atccgccagg gctacggcct gacagaaaca accagcgcca ttctgatcac ccccgaaggg
     1441 gacgacaagc ctggcgcagt aggcaaggtg gtgcccttct tcgaggctaa ggtggtggac
     1501 ttggacaccg gtaagacact gggtgtgaac cagcgcggcg agctgtgcgt ccgtggcccc
     1561 atgatcatga gcggctacgt taacaacccc gaggctacaa acgctctcat cgacaaggac
     1621 ggctggctgc acagcggcga catcgcctac tgggacgagg acgagcactt cttcatcgtg
     1681 gaccggctga agagcctgat caaatacaag ggctaccagg tagccccagc cgaactggag
     1741 agcatcctgc tgcaacaccc caacatcttc gacgccgggg tcgccggcct gcccgacgac
     1801 gatgccggcg agctgcccgc cgcagtcgtc gtgctggaac acggtaaaac catgaccgag
     1861 aaggagatcg tggactatgt ggccagccag gttacaaccg ccaagaagct gcgcggtggt
     1921 gttgtgttcg tggacgaggt gcctaaagga ctgaccggca agttggacgc ccgcaagatc
     1981 cgcgagattc tcattaaggc caagaaggga tccaacgtgg tggtgcacca ggccggcggt
     2041 gccaaaaaca ttaagaaggg cccagcgcca ttctacccac tcgaagacgg gaccgccggc
     2101 gagcagctgc acaaagccat gaagcgctac gccctggtgc ccggcaccat cgcctttacc
     2161 gacgcacata tcgaggtgga cattacctac gccgagtact tcgagatgag cgttcggctg
     2221 gcagaagcta tgaagcgcta tgggctgaat acaaaccatc ggatcgtggt gtgcagcgag
     2281 aatagcttgc agttcttcat gcccgtgttg ggtgccctgt tcatcggtgt ggctgtggcc
     2341 ccagctaacg acatctacaa cgagcgcgag ctgctgaaca gcatgggcat cagccagccc
     2401 accgtcgtat tcgtgagcaa gaaagggctg caaaagatcc tcaacgtgca aaagaagcta
     2461 ccgatcatac aaaagatcat catcatggat agcaagaccg actaccaggg cttccaaagc
     2521 atgtacacct tcgtgacttc ccatttgcca cccggcttca acgagtacga cttcgtgccc
     2581 gagagcttcg accgggacaa aaccatcgcc ctgatcatga acagtagtgg cagtaccgga
     2641 ttgcccaagg gcgtagccct accgcaccgc accgcttgtg tccgattcag tcatgcccgc
     2701 gaccccatct tcggcaacca gatcatcccc gccgagtact gtttaagcta tgaaacggaa
     2761 atattgacag tagaatatgg attattaccg attggtaaaa ttgtagaaaa gcgcatcgaa
     2821 tgtactgttt atagcgttga taataatgga aatatttata cacaacctgt agcacaatgg
     2881 cacgatcgcg gagaacaaga ggtgtttgag tattgtttgg aagatggttc attgattcgg
     2941 gcaacaaaag accataagtt tatgactgtt gatggtcaaa tgttgccaat tgatgaaata
     3001 tttgaacgtg aattggattt gatgcgggtt gataatttgc cgaacggcgg caagatcgcc
     3061 gtcaattctg cttgcaagaa ctggttcagt agcttaagcc actttgtgat ccaccttaac
     3121 agccacggct tccctcccga ggtggaggag caggccgccg gcaccctgcc catgagctgc
     3181 gcccaggaga gcggcatgga tagacaccct gctgcttgcg ccagcgccag gatcaacgtc
     3241 taactgcagt ctagagggcc ctattctata gtgtcaccta aatgctagag ctcgctgatc
     3301 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     3361 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     3421 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     3481 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     3541 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     3601 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     3661 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     3721 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     3781 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     3841 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     3901 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     3961 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     4021 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     4081 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     4141 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     4201 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4261 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     4321 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     4381 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     4441 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     4501 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     4561 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     4621 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     4681 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     4741 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     4801 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     4861 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     4921 gaactgttcg ctaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     4981 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     5041 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     5101 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     5161 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     5221 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     5281 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     5341 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     5401 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     5461 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     5521 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     5581 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     5641 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     5701 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     5761 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     5821 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     5881 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     5941 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     6001 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     6061 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     6121 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     6181 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     6241 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     6301 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     6361 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     6421 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     6481 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
     6541 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac gaaaactcac
     6601 gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc cttttaaatt
     6661 aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct gacagttacc
     6721 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
     6781 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
     6841 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
     6901 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
     6961 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
     7021 ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
     7081 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
     7141 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     7201 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     7261 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     7321 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     7381 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     7441 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     7501 ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
     7561 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
     7621 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata ggggttccgc
     7681 gcacatttcc ccgaaaagtg ccacctgacg tc
//
