LOCUS       sequence-486451-        8333 bp    DNA     circular SYN 18-SEP-2025
DEFINITION  Encodes for mammalian expression of TVMV, TEV and HRV-3C 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 8333)
  TITLE     Boolean Logic-gated Protein Presentation via Autonomously Compiled 
            Molecular Topology
REFERENCE   2  (bases 1 to 8333)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Sep 18, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: TVMV-TEV-HRV-3C Proteases-mCherry
FEATURES             Location/Qualifiers
     source          1..8333
                     /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"
     CDS             3077..3133
                     /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     3750..3773
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3924..3941)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3930..4154
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4200..4628
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4287..4306)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4497..4518
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(4637..4657)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        4642..4971
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4822..4957
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4884..4903
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5038..5832
                     /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(5092..5111)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5702..5721
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6006..6127
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6043..6062)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6097..6116
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6176..6192)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6176..6192)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6189..6211)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6200..6216
                     /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(6224..6230,6231..6248,6249..6254))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6269..6290
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6407..6424)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6578..7166)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6658..6677)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(7337..8128,8129..8197))
                     /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     7960..7979
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8198..8302)
                     /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 gcggctcggg
     2521 acctaacacc gagttcgccc tgagcctact gagaaagaac atcatgacca tcaccaccag
     2581 caagggcgag ttcaccggcc tgggcatcca cgacagagtg tgcgtgatcc ccacccatgc
     2641 ccagccggga gatgatgtgt tggtgaacgg ccaaaaaatc cgggtgaagg acaagtacaa
     2701 gctggtggac cctgaaaaca tcaacctgga actgaccgtg ctgacactgg atcggaatga
     2761 aaaatttcga gacatcagag gctttatctc tgaggacctg gaaggcgtcg acgccacact
     2821 ggtggtccac agcaacaact ttacaaatac catcttggag gtgggccctg tgaccatggc
     2881 tggcctgatc aacctgtcta gcacacctac aaacagaatg atccgatacg actacgccac
     2941 aaaaaccggc cagtgtggcg gagtgctgtg cgccactggc aaaattttcg gcattcacgt
     3001 gggaggcaat ggcagacagg gcttctctgc ccagctgaag aagcagtact tcgtggaaaa
     3061 gcagggctcc ggcagtgcca caaatttcag cctgctgaag caggccggcg acgtggaaga
     3121 gaaccctggc cccgggagcg gctccgtgag caagggcgag gaagataaca tggctatcat
     3181 caaggagttc atgagattca aggtgcacat ggaaggcagc gtgaacggtc acgagtttga
     3241 gatcgagggc gaaggcgaag gcagacccta cgagggaaca cagaccgcca agctgaaagt
     3301 gacaaagggc ggacctctgc cattcgcctg ggacatcctg agccctcagt tcatgtatgg
     3361 cagcaaggca tatgtgaagc accccgccga tatccctgac tacctgaaac tgtcttttcc
     3421 tgagggattc aagtgggagc gtgtgatgaa cttcgaggac ggcggggtcg tgaccgtgac
     3481 ccaggacagc tccctccagg acggcgagtt catctacaag gtgaagctga ggggcacaaa
     3541 cttcccctct gacggccccg tgatgcagaa gaaaacaatg ggctgggagg ccagctcaga
     3601 aagaatgtac cctgaggacg gagccctgaa aggcgaaatc aaacaaagac tgaagctgaa
     3661 ggatggaggc cactacgacg ccgaagtcaa gacaacctac aaggccaaaa agcctgtgca
     3721 gctgcctggc gcctacaacg tgaatatcaa gctggacatc acctcccaca acgaggacta
     3781 caccatcgtg gagcagtacg agcgggccga gggcagacac agcaccggcg gcatggacga
     3841 gctgtacaag tgagaattct gcagatatcc agcacagtgg cggccgctcg agtctagagg
     3901 gcccgtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc catctgttgt
     3961 ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg tcctttccta
     4021 ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc tggggggtgg
     4081 ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg ctggggatgc
     4141 ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg ggtatcccca
     4201 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     4261 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     4321 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     4381 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     4441 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     4501 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     4561 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     4621 cgcgaattaa ttctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     4681 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     4741 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     4801 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     4861 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc tgcctctgag
     4921 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa aaagctcccg
     4981 ggagcttgta tatccatttt cggatctgat caagagacag gatgaggatc gtttcgcatg
     5041 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     5101 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     5161 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcag
     5221 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     5281 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     5341 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     5401 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     5461 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagag
     5521 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgcgcat gcccgacggc
     5581 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     5641 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     5701 gcgttggcta cccgtgatat tgctgaagag cttggcggcg aatgggctga ccgcttcctc
     5761 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     5821 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     5881 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     5941 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     6001 accccaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     6061 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     6121 tatcttatca tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat
     6181 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     6241 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     6301 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc
     6361 aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact
     6421 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     6481 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     6541 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     6601 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     6661 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     6721 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     6781 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     6841 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     6901 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     6961 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa
     7021 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     7081 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     7141 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     7201 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct
     7261 tcacctagat ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt
     7321 aaacttggtc tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc
     7381 tatttcgttc atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg
     7441 gcttaccatc tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag
     7501 atttatcagc aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt
     7561 tatccgcctc catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag
     7621 ttaatagttt gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt
     7681 ttggtatggc ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca
     7741 tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg
     7801 ccgcagtgtt atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat
     7861 ccgtaagatg cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta
     7921 tgcggcgacc gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca
     7981 gaactttaaa agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct
     8041 taccgctgtt gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat
     8101 cttttacttt caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa
     8161 agggaataag ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt
     8221 gaagcattta tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa
     8281 ataaacaaat aggggttccg cgcacatttc cccgaaaagt gccacctgac gtc
//
