LOCUS       sequence-486450-        6902 bp    DNA     circular SYN 10-OCT-2025
DEFINITION  Encodes for mammalian expression of TVMV 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 6902)
  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 6902)
  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: TVMV Protease-mCherry
FEATURES             Location/Qualifiers
     source          1..6902
                     /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             929..1633
                     /codon_start=1
                     /product="tobacco vein mottling virus NIa protease 
                     (Nallamsetty et al., 2004)"
                     /label=TVMV protease
                     /translation="SKALLKGVRDFNPISACVCLLENSSDGHSERLFGIGFGPYIIANQ
                     HLFRRNNGELTIKTMHGEFKVKNSTQLQMKPVEGRDIIVIKMAKDFPPFPQKLKFRQPT
                     IKDRVCMVSTNFQQKSVSSLVSESSHIVHKEDTSFWQHWITTKDGQCGSPLVSIIDGNI
                     LGIHSLTHTTNGSNYFVEFPEKFVATYLDAADGWCKNWKFNADKISWGSFTLVEDAPED
                     DFMAKKTVAAIMD"
     CDS             1646..1702
                     /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     2319..2342
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2493..2510)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2499..2723
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2769..3197
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2856..2875)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3066..3087
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3206..3226)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3211..3540
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3391..3526
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3453..3472
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3607..4401
                     /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(3661..3680)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4271..4290
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4575..4696
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4612..4631)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4666..4685
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4745..4761)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4745..4761)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4758..4780)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4769..4785
                     /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(4793..4799,4800..4817,4818..4823))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4838..4859
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4976..4993)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5147..5735)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5227..5246)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5906..6697,6698..6766))
                     /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     6529..6548
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6767..6871)
                     /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 gctcctctag caaggctctg ctgaaaggag tgcgggattt
      961 caaccccatc agcgcatgcg tgtgcctgct ggagaacagc tctgacggac actctgaaag
     1021 actcttcggc atcggcttcg gcccttacat catcgccaat cagcaccttt tccggagaaa
     1081 caacggcgag ctgacaatca agaccatgca cggagagttc aaggtgaaga atagtacaca
     1141 gctgcagatg aaacccgttg agggacggga catcatcgtg atcaagatgg ctaaggactt
     1201 ccctccattc ccccagaaac tgaagttccg gcagcctacc atcaaggata gagtgtgtat
     1261 ggtgagcacc aacttccagc agaagagcgt atccagcctg gtgtccgaaa gcagtcacat
     1321 cgtgcataag gaagacacct ccttctggca gcactggatc accaccaagg acggacaatg
     1381 tggctctcct ctggtgagca tcattgacgg caacatcctg ggcatccact ctctcacaca
     1441 taccacaaat gggagcaact acttcgtgga gttccctgaa aagtttgtgg ccacgtacct
     1501 ggatgccgct gacggctggt gcaagaattg gaagttcaac gccgataaga tcagctgggg
     1561 cagcttcacc ctggtggagg atgcccctga ggatgacttc atggccaaga agaccgtcgc
     1621 cgccatcatg gacggtagcg gcagcgccac aaacttcagc ctcctcaagc aggctgggga
     1681 cgttgaggag aaccctggcc ccggctccgg ctctgtgagc aagggcgagg aagataacat
     1741 ggctatcatc aaggagttca tgagattcaa ggtgcacatg gaaggcagcg tgaacggtca
     1801 cgagtttgag atcgagggcg aaggcgaagg cagaccctac gagggaacac agaccgccaa
     1861 gctgaaagtg acaaagggcg gacctctgcc attcgcctgg gacatcctga gccctcagtt
     1921 catgtatggc agcaaggcat atgtgaagca ccccgccgat atccctgact acctgaaact
     1981 gtcttttcct gagggattca agtgggagcg tgtgatgaac ttcgaggacg gcggggtcgt
     2041 gaccgtgacc caggacagct ccctccagga cggcgagttc atctacaagg tgaagctgag
     2101 gggcacaaac ttcccctctg acggccccgt gatgcagaag aaaacaatgg gctgggaggc
     2161 cagctcagaa agaatgtacc ctgaggacgg agccctgaaa ggcgaaatca aacaaagact
     2221 gaagctgaag gatggaggcc actacgacgc cgaagtcaag acaacctaca aggccaaaaa
     2281 gcctgtgcag ctgcctggcg cctacaacgt gaatatcaag ctggacatca cctcccacaa
     2341 cgaggactac accatcgtgg agcagtacga gcgggccgag ggcagacaca gcaccggcgg
     2401 catggacgag ctgtacaagt gagaattctg cagatatcca gcacagtggc ggccgctcga
     2461 gtctagaggg cccgtttaaa cccgctgatc agcctcgact gtgccttcta gttgccagcc
     2521 atctgttgtt tgcccctccc ccgtgccttc cttgaccctg gaaggtgcca ctcccactgt
     2581 cctttcctaa taaaatgagg aaattgcatc gcattgtctg agtaggtgtc attctattct
     2641 ggggggtggg gtggggcagg acagcaaggg ggaggattgg gaagacaata gcaggcatgc
     2701 tggggatgcg gtgggctcta tggcttctga ggcggaaaga accagctggg gctctagggg
     2761 gtatccccac gcgccctgta gcggcgcatt aagcgcggcg ggtgtggtgg ttacgcgcag
     2821 cgtgaccgct acacttgcca gcgccctagc gcccgctcct ttcgctttct tcccttcctt
     2881 tctcgccacg ttcgccggct ttccccgtca agctctaaat cgggggctcc ctttagggtt
     2941 ccgatttagt gctttacggc acctcgaccc caaaaaactt gattagggtg atggttcacg
     3001 tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt ccacgttctt
     3061 taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg tctattcttt
     3121 tgatttataa gggattttgc cgatttcggc ctattggtta aaaaatgagc tgatttaaca
     3181 aaaatttaac gcgaattaat tctgtggaat gtgtgtcagt tagggtgtgg aaagtcccca
     3241 ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc aaccaggtgt
     3301 ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct caattagtca
     3361 gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc cagttccgcc
     3421 cattctccgc cccatggctg actaattttt tttatttatg cagaggccga ggccgcctct
     3481 gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg cttttgcaaa
     3541 aagctcccgg gagcttgtat atccattttc ggatctgatc aagagacagg atgaggatcg
     3601 tttcgcatga ttgaacaaga tggattgcac gcaggttctc cggccgcttg ggtggagagg
     3661 ctattcggct atgactgggc acaacagaca atcggctgct ctgatgccgc cgtgttccgg
     3721 ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg acctgtccgg tgccctgaat
     3781 gaactgcagg acgaggcagc gcggctatcg tggctggcca cgacgggcgt tccttgcgca
     3841 gctgtgctcg acgttgtcac tgaagcggga agggactggc tgctattggg cgaagtgccg
     3901 gggcaggatc tcctgtcatc tcaccttgct cctgccgaga aagtatccat catggctgat
     3961 gcaatgcggc ggctgcatac gcttgatccg gctacctgcc cattcgacca ccaagcgaaa
     4021 catcgcatcg agcgagcacg tactcggatg gaagccggtc ttgtcgatca ggatgatctg
     4081 gacgaagagc atcaggggct cgcgccagcc gaactgttcg ccaggctcaa ggcgcgcatg
     4141 cccgacggcg aggatctcgt cgtgacccat ggcgatgcct gcttgccgaa tatcatggtg
     4201 gaaaatggcc gcttttctgg attcatcgac tgtggccggc tgggtgtggc ggaccgctat
     4261 caggacatag cgttggctac ccgtgatatt gctgaagagc ttggcggcga atgggctgac
     4321 cgcttcctcg tgctttacgg tatcgccgct cccgattcgc agcgcatcgc cttctatcgc
     4381 cttcttgacg agttcttctg agcgggactc tggggttcga aatgaccgac caagcgacgc
     4441 ccaacctgcc atcacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     4501 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     4561 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     4621 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     4681 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     4741 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4801 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4861 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     4921 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     4981 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5041 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5101 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5161 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5221 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5281 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5341 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5401 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5461 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5521 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5581 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5641 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5701 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5761 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5821 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5881 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5941 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6001 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6061 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6121 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6181 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6241 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6301 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6361 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6421 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6481 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6541 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6601 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6661 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6721 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6781 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6841 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6901 tc
//
