LOCUS       sequence-486454-        7547 bp    DNA     circular SYN 10-OCT-2025
DEFINITION  Encodes for mammalian expression of 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 7547)
  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 7547)
  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: TEV-HRV-3C Proteases-mCherry
FEATURES             Location/Qualifiers
     source          1..7547
                     /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             1664..1720
                     /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             2291..2347
                     /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     2964..2987
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(3138..3155)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3144..3368
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3414..3842
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3501..3520)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3711..3732
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3851..3871)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3856..4185
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      4036..4171
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     4098..4117
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             4252..5046
                     /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(4306..4325)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4916..4935
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5220..5341
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5257..5276)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5311..5330
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5390..5406)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5390..5406)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5403..5425)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5414..5430
                     /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(5438..5444,5445..5462,5463..5468))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5483..5504
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5621..5638)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5792..6380)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5872..5891)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6551..7342,7343..7411))
                     /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     7174..7193
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7412..7516)
                     /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 ggagagcctg tttaaaggcc ccagagatta
      961 taatcccatc agctccacca tcgtgcacct gacaaacgag agcgacggac acaccacaag
     1021 cctgtacgga atcggctttg gacctttcat cattaccaac aagcacctgt tcagacggaa
     1081 caacggcacg ctggtggtgc agagcctgca cggcgttttt aaggtgaaga acaccaccac
     1141 cctgcagcag catctgatcg acggaagaga catgatcatc atcaggatgc ctaaggactt
     1201 cccacctttc cctcagaagc tgaaattcag ggagcctcag agagaggagc ggatcgtcct
     1261 ggtgaccacc aacttccaga caaaaagcat gagctctatg gtgtccgata caagcagcac
     1321 cttcccttcc ggcgacggca tcttctggaa gcactggatc caaaccaaag atggccagtg
     1381 cggctctccc ctggtgagca ctagagatgg cttcatcgtg ggcatccaca gcgctagcaa
     1441 cttcaccaac actaacaact actttaccag cgttcctaag aatttcatgg aactgctgac
     1501 caaccaggaa gcccaacaat gggtgagcgg ttggcggctg aacgccgatt ctgtgctgtg
     1561 gggaggacac aaggtattca tggataagcc tgaagaacct ttccagcctg tcaaagaggc
     1621 cacacagctg atgaaccgga gaagaagaag aggcagcggc agcgccacca atttcagcct
     1681 gctgaagcag gctggcgacg tggaggaaaa cccaggccca ggtagcggct cgggacctaa
     1741 caccgagttc gccctgagcc tactgagaaa gaacatcatg accatcacca ccagcaaggg
     1801 cgagttcacc ggcctgggca tccacgacag agtgtgcgtg atccccaccc atgcccagcc
     1861 gggagatgat gtgttggtga acggccaaaa aatccgggtg aaggacaagt acaagctggt
     1921 ggaccctgaa aacatcaacc tggaactgac cgtgctgaca ctggatcgga atgaaaaatt
     1981 tcgagacatc agaggcttta tctctgagga cctggaaggc gtcgacgcca cactggtggt
     2041 ccacagcaac aactttacaa ataccatctt ggaggtgggc cctgtgacca tggctggcct
     2101 gatcaacctg tctagcacac ctacaaacag aatgatccga tacgactacg ccacaaaaac
     2161 cggccagtgt ggcggagtgc tgtgcgccac tggcaaaatt ttcggcattc acgtgggagg
     2221 caatggcaga cagggcttct ctgcccagct gaagaagcag tacttcgtgg aaaagcaggg
     2281 ctccggcagt gccacaaatt tcagcctgct gaagcaggcc ggcgacgtgg aagagaaccc
     2341 tggccccggg agcggctccg tgagcaaggg cgaggaagat aacatggcta tcatcaagga
     2401 gttcatgaga ttcaaggtgc acatggaagg cagcgtgaac ggtcacgagt ttgagatcga
     2461 gggcgaaggc gaaggcagac cctacgaggg aacacagacc gccaagctga aagtgacaaa
     2521 gggcggacct ctgccattcg cctgggacat cctgagccct cagttcatgt atggcagcaa
     2581 ggcatatgtg aagcaccccg ccgatatccc tgactacctg aaactgtctt ttcctgaggg
     2641 attcaagtgg gagcgtgtga tgaacttcga ggacggcggg gtcgtgaccg tgacccagga
     2701 cagctccctc caggacggcg agttcatcta caaggtgaag ctgaggggca caaacttccc
     2761 ctctgacggc cccgtgatgc agaagaaaac aatgggctgg gaggccagct cagaaagaat
     2821 gtaccctgag gacggagccc tgaaaggcga aatcaaacaa agactgaagc tgaaggatgg
     2881 aggccactac gacgccgaag tcaagacaac ctacaaggcc aaaaagcctg tgcagctgcc
     2941 tggcgcctac aacgtgaata tcaagctgga catcacctcc cacaacgagg actacaccat
     3001 cgtggagcag tacgagcggg ccgagggcag acacagcacc ggcggcatgg acgagctgta
     3061 caagtgagaa ttctgcagat atccagcaca gtggcggccg ctcgagtcta gagggcccgt
     3121 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     3181 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     3241 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     3301 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     3361 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     3421 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     3481 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     3541 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     3601 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     3661 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     3721 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     3781 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     3841 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     3901 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     3961 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     4021 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     4081 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     4141 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     4201 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     4261 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     4321 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     4381 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     4441 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     4501 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     4561 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     4621 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     4681 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     4741 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     4801 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     4861 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     4921 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     4981 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     5041 ttctgagcgg gactctgggg ttcgaaatga ccgaccaagc gacgcccaac ctgccatcac
     5101 gagatttcga ttccaccgcc gccttctatg aaaggttggg cttcggaatc gttttccggg
     5161 acgccggctg gatgatcctc cagcgcgggg atctcatgct ggagttcttc gcccacccca
     5221 acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa
     5281 ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt
     5341 atcatgtctg tataccgtcg acctctagct agagcttggc gtaatcatgg tcatagctgt
     5401 ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc ggaagcataa
     5461 agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg ttgcgctcac
     5521 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5581 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     5641 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     5701 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5761 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5821 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5881 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5941 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     6001 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     6061 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     6121 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     6181 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     6241 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     6301 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     6361 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     6421 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     6481 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     6541 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     6601 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     6661 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6721 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6781 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6841 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6901 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6961 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     7021 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     7081 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     7141 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     7201 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     7261 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     7321 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     7381 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     7441 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     7501 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtc
//
