LOCUS       sequence-486448-        7706 bp    DNA     circular SYN 10-OCT-2025
DEFINITION  Encodes for mammalian expression of TVMV and TEV protease inputs. 
            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 7706)
  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 7706)
  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-TEV Proteases-mCherry
FEATURES             Location/Qualifiers
     source          1..7706
                     /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"
     CDS             2450..2506
                     /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     3123..3146
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3297..3314)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3303..3527
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3573..4001
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3660..3679)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3870..3891
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4010..4030)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4015..4344
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4195..4330
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4257..4276
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4411..5205
                     /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(4465..4484)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5075..5094
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5379..5500
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5416..5435)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5470..5489
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5549..5565)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5549..5565)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5562..5584)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5573..5589
                     /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(5597..5603,5604..5621,5622..5627))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5642..5663
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5780..5797)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5951..6539)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6031..6050)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6710..7501,7502..7570))
                     /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     7333..7352
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7571..7675)
                     /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 ctctggggag agcctgttta aaggccccag
     1741 agattataat cccatcagct ccaccatcgt gcacctgaca aacgagagcg acggacacac
     1801 cacaagcctg tacggaatcg gctttggacc tttcatcatt accaacaagc acctgttcag
     1861 acggaacaac ggcacgctgg tggtgcagag cctgcacggc gtttttaagg tgaagaacac
     1921 caccaccctg cagcagcatc tgatcgacgg aagagacatg atcatcatca ggatgcctaa
     1981 ggacttccca cctttccctc agaagctgaa attcagggag cctcagagag aggagcggat
     2041 cgtcctggtg accaccaact tccagacaaa aagcatgagc tctatggtgt ccgatacaag
     2101 cagcaccttc ccttccggcg acggcatctt ctggaagcac tggatccaaa ccaaagatgg
     2161 ccagtgcggc tctcccctgg tgagcactag agatggcttc atcgtgggca tccacagcgc
     2221 tagcaacttc accaacacta acaactactt taccagcgtt cctaagaatt tcatggaact
     2281 gctgaccaac caggaagccc aacaatgggt gagcggttgg cggctgaacg ccgattctgt
     2341 gctgtgggga ggacacaagg tattcatgga taagcctgaa gaacctttcc agcctgtcaa
     2401 agaggccaca cagctgatga accggagaag aagaagaggc agcggcagcg ccaccaattt
     2461 cagcctgctg aagcaggctg gcgacgtgga ggaaaaccca ggcccaggta gcggctcggt
     2521 gagcaagggc gaggaagata acatggctat catcaaggag ttcatgagat tcaaggtgca
     2581 catggaaggc agcgtgaacg gtcacgagtt tgagatcgag ggcgaaggcg aaggcagacc
     2641 ctacgaggga acacagaccg ccaagctgaa agtgacaaag ggcggacctc tgccattcgc
     2701 ctgggacatc ctgagccctc agttcatgta tggcagcaag gcatatgtga agcaccccgc
     2761 cgatatccct gactacctga aactgtcttt tcctgaggga ttcaagtggg agcgtgtgat
     2821 gaacttcgag gacggcgggg tcgtgaccgt gacccaggac agctccctcc aggacggcga
     2881 gttcatctac aaggtgaagc tgaggggcac aaacttcccc tctgacggcc ccgtgatgca
     2941 gaagaaaaca atgggctggg aggccagctc agaaagaatg taccctgagg acggagccct
     3001 gaaaggcgaa atcaaacaaa gactgaagct gaaggatgga ggccactacg acgccgaagt
     3061 caagacaacc tacaaggcca aaaagcctgt gcagctgcct ggcgcctaca acgtgaatat
     3121 caagctggac atcacctccc acaacgagga ctacaccatc gtggagcagt acgagcgggc
     3181 cgagggcaga cacagcaccg gcggcatgga cgagctgtac aagtgagaat tctgcagata
     3241 tccagcacag tggcggccgc tcgagtctag agggcccgtt taaacccgct gatcagcctc
     3301 gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc cttccttgac
     3361 cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg catcgcattg
     3421 tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca agggggagga
     3481 ttgggaagac aatagcaggc atgctgggga tgcggtgggc tctatggctt ctgaggcgga
     3541 aagaaccagc tggggctcta gggggtatcc ccacgcgccc tgtagcggcg cattaagcgc
     3601 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
     3661 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
     3721 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
     3781 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
     3841 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
     3901 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
     3961 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat taattctgtg gaatgtgtgt
     4021 cagttagggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca aagcatgcat
     4081 ctcaattagt cagcaaccag gtgtggaaag tccccaggct ccccagcagg cagaagtatg
     4141 caaagcatgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc gcccatcccg
     4201 cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat tttttttatt
     4261 tatgcagagg ccgaggccgc ctctgcctct gagctattcc agaagtagtg aggaggcttt
     4321 tttggaggcc taggcttttg caaaaagctc ccgggagctt gtatatccat tttcggatct
     4381 gatcaagaga caggatgagg atcgtttcgc atgattgaac aagatggatt gcacgcaggt
     4441 tctccggccg cttgggtgga gaggctattc ggctatgact gggcacaaca gacaatcggc
     4501 tgctctgatg ccgccgtgtt ccggctgtca gcgcaggggc gcccggttct ttttgtcaag
     4561 accgacctgt ccggtgccct gaatgaactg caggacgagg cagcgcggct atcgtggctg
     4621 gccacgacgg gcgttccttg cgcagctgtg ctcgacgttg tcactgaagc gggaagggac
     4681 tggctgctat tgggcgaagt gccggggcag gatctcctgt catctcacct tgctcctgcc
     4741 gagaaagtat ccatcatggc tgatgcaatg cggcggctgc atacgcttga tccggctacc
     4801 tgcccattcg accaccaagc gaaacatcgc atcgagcgag cacgtactcg gatggaagcc
     4861 ggtcttgtcg atcaggatga tctggacgaa gagcatcagg ggctcgcgcc agccgaactg
     4921 ttcgccaggc tcaaggcgcg catgcccgac ggcgaggatc tcgtcgtgac ccatggcgat
     4981 gcctgcttgc cgaatatcat ggtggaaaat ggccgctttt ctggattcat cgactgtggc
     5041 cggctgggtg tggcggaccg ctatcaggac atagcgttgg ctacccgtga tattgctgaa
     5101 gagcttggcg gcgaatgggc tgaccgcttc ctcgtgcttt acggtatcgc cgctcccgat
     5161 tcgcagcgca tcgccttcta tcgccttctt gacgagttct tctgagcggg actctggggt
     5221 tcgaaatgac cgaccaagcg acgcccaacc tgccatcacg agatttcgat tccaccgccg
     5281 ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg atgatcctcc
     5341 agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt gcagcttata
     5401 atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt ttttcactgc
     5461 attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt ataccgtcga
     5521 cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     5581 cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct
     5641 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     5701 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     5761 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     5821 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     5881 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     5941 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     6001 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     6061 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     6121 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     6181 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     6241 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     6301 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     6361 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     6421 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     6481 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     6541 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     6601 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     6661 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     6721 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     6781 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     6841 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     6901 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     6961 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     7021 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     7081 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     7141 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     7201 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     7261 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     7321 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     7381 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     7441 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     7501 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     7561 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     7621 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     7681 ttccccgaaa agtgccacct gacgtc
//
