LOCUS       sequence-486453-        6743 bp    DNA     circular SYN 10-OCT-2025
DEFINITION  Encodes for mammalian expression of HRV-3C protease input. Construct
            contains a mCherry to indicate successful transfection and full 
            plasmid read-through. Each protein is separated by a P2A 
            self-cleaving sequence..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6743)
  AUTHORS   Gharios R, Ross ML, Li A, Kottantharayil SP, Hoye J, DeForest CA
  TITLE     Boolean logic-gated protein presentation through autonomously 
            compiled molecular topology.
  JOURNAL   Nat Chem Biol. 2025 Oct 9. doi: 10.1038/s41589-025-02037-5.
  PUBMED    41068299
REFERENCE   2  (bases 1 to 6743)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 10, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nat Chem 
            Biol. 2025 Oct 9. doi: 10.1038/s41589-025-02037-5."
COMMENT     Sequence Label: HRV-3C Protease-mCherry
FEATURES             Location/Qualifiers
     source          1..6743
                     /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             1487..1543
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     primer_bind     2160..2183
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2334..2351)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2340..2564
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2610..3038
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2697..2716)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2907..2928
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3047..3067)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3052..3381
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3232..3367
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3294..3313
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3448..4242
                     /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(3502..3521)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4112..4131
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4416..4537
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4453..4472)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4507..4526
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4586..4602)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4586..4602)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4599..4621)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4610..4626
                     /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(4634..4640,4641..4658,4659..4664))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4679..4700
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4817..4834)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4988..5576)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5068..5087)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5747..6538,6539..6607))
                     /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     6370..6389
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6608..6712)
                     /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 gctggctagc
      901 gtttaaactt aagcttatgg gctcctctgg acctaacacc gagttcgccc tgagcctact
      961 gagaaagaac atcatgacca tcaccaccag caagggcgag ttcaccggcc tgggcatcca
     1021 cgacagagtg tgcgtgatcc ccacccatgc ccagccggga gatgatgtgt tggtgaacgg
     1081 ccaaaaaatc cgggtgaagg acaagtacaa gctggtggac cctgaaaaca tcaacctgga
     1141 actgaccgtg ctgacactgg atcggaatga aaaatttcga gacatcagag gctttatctc
     1201 tgaggacctg gaaggcgtcg acgccacact ggtggtccac agcaacaact ttacaaatac
     1261 catcttggag gtgggccctg tgaccatggc tggcctgatc aacctgtcta gcacacctac
     1321 aaacagaatg atccgatacg actacgccac aaaaaccggc cagtgtggcg gagtgctgtg
     1381 cgccactggc aaaattttcg gcattcacgt gggaggcaat ggcagacagg gcttctctgc
     1441 ccagctgaag aagcagtact tcgtggaaaa gcagggctcc ggcagtgcca caaatttcag
     1501 cctgctgaag caggccggcg acgtggaaga gaaccctggc cccgggagcg gctccgtgag
     1561 caagggcgag gaagataaca tggctatcat caaggagttc atgagattca aggtgcacat
     1621 ggaaggcagc gtgaacggtc acgagtttga gatcgagggc gaaggcgaag gcagacccta
     1681 cgagggaaca cagaccgcca agctgaaagt gacaaagggc ggacctctgc cattcgcctg
     1741 ggacatcctg agccctcagt tcatgtatgg cagcaaggca tatgtgaagc accccgccga
     1801 tatccctgac tacctgaaac tgtcttttcc tgagggattc aagtgggagc gtgtgatgaa
     1861 cttcgaggac ggcggggtcg tgaccgtgac ccaggacagc tccctccagg acggcgagtt
     1921 catctacaag gtgaagctga ggggcacaaa cttcccctct gacggccccg tgatgcagaa
     1981 gaaaacaatg ggctgggagg ccagctcaga aagaatgtac cctgaggacg gagccctgaa
     2041 aggcgaaatc aaacaaagac tgaagctgaa ggatggaggc cactacgacg ccgaagtcaa
     2101 gacaacctac aaggccaaaa agcctgtgca gctgcctggc gcctacaacg tgaatatcaa
     2161 gctggacatc acctcccaca acgaggacta caccatcgtg gagcagtacg agcgggccga
     2221 gggcagacac agcaccggcg gcatggacga gctgtacaag tgagaattct gcagatatcc
     2281 agcacagtgg cggccgctcg agtctagagg gcccgtttaa acccgctgat cagcctcgac
     2341 tgtgccttct agttgccagc catctgttgt ttgcccctcc cccgtgcctt ccttgaccct
     2401 ggaaggtgcc actcccactg tcctttccta ataaaatgag gaaattgcat cgcattgtct
     2461 gagtaggtgt cattctattc tggggggtgg ggtggggcag gacagcaagg gggaggattg
     2521 ggaagacaat agcaggcatg ctggggatgc ggtgggctct atggcttctg aggcggaaag
     2581 aaccagctgg ggctctaggg ggtatcccca cgcgccctgt agcggcgcat taagcgcggc
     2641 gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc
     2701 tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc aagctctaaa
     2761 tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc ccaaaaaact
     2821 tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt ttcgcccttt
     2881 gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa caacactcaa
     2941 ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg cctattggtt
     3001 aaaaaatgag ctgatttaac aaaaatttaa cgcgaattaa ttctgtggaa tgtgtgtcag
     3061 ttagggtgtg gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc
     3121 aattagtcag caaccaggtg tggaaagtcc ccaggctccc cagcaggcag aagtatgcaa
     3181 agcatgcatc tcaattagtc agcaaccata gtcccgcccc taactccgcc catcccgccc
     3241 ctaactccgc ccagttccgc ccattctccg ccccatggct gactaatttt ttttatttat
     3301 gcagaggccg aggccgcctc tgcctctgag ctattccaga agtagtgagg aggctttttt
     3361 ggaggcctag gcttttgcaa aaagctcccg ggagcttgta tatccatttt cggatctgat
     3421 caagagacag gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct
     3481 ccggccgctt gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc
     3541 tctgatgccg ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc
     3601 gacctgtccg gtgccctgaa tgaactgcag gacgaggcag cgcggctatc gtggctggcc
     3661 acgacgggcg ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg
     3721 ctgctattgg gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag
     3781 aaagtatcca tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc
     3841 ccattcgacc accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt
     3901 cttgtcgatc aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc
     3961 gccaggctca aggcgcgcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc
     4021 tgcttgccga atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg
     4081 ctgggtgtgg cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag
     4141 cttggcggcg aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg
     4201 cagcgcatcg ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg
     4261 aaatgaccga ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct
     4321 tctatgaaag gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc
     4381 gcggggatct catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg
     4441 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     4501 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct
     4561 ctagctagag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     4621 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4681 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4741 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     4801 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4861 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4921 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4981 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     5041 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     5101 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     5161 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     5221 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     5281 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     5341 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     5401 ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc
     5461 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     5521 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     5581 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     5641 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     5701 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     5761 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5821 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5881 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5941 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     6001 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     6061 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     6121 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     6181 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     6241 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     6301 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     6361 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     6421 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     6481 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     6541 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     6601 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     6661 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     6721 cccgaaaagt gccacctgac gtc
//
