LOCUS       sequence-486452-        6920 bp    DNA     circular SYN 10-OCT-2025
DEFINITION  Encodes for mammalian expression of TEV 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 6920)
  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 6920)
  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 Protease-mCherry
FEATURES             Location/Qualifiers
     source          1..6920
                     /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"
     primer_bind     2337..2360
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     primer_bind     complement(2511..2528)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2517..2741
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      2787..3215
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2874..2893)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3084..3105
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3224..3244)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3229..3558
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3409..3544
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3471..3490
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             3625..4419
                     /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(3679..3698)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4289..4308
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    4593..4714
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4630..4649)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4684..4703
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4763..4779)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4763..4779)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4776..4798)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4787..4803
                     /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(4811..4817,4818..4835,4836..4841))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4856..4877
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4994..5011)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5165..5753)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5245..5264)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5924..6715,6716..6784))
                     /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     6547..6566
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6785..6889)
                     /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 cggtgagcaa
     1741 gggcgaggaa gataacatgg ctatcatcaa ggagttcatg agattcaagg tgcacatgga
     1801 aggcagcgtg aacggtcacg agtttgagat cgagggcgaa ggcgaaggca gaccctacga
     1861 gggaacacag accgccaagc tgaaagtgac aaagggcgga cctctgccat tcgcctggga
     1921 catcctgagc cctcagttca tgtatggcag caaggcatat gtgaagcacc ccgccgatat
     1981 ccctgactac ctgaaactgt cttttcctga gggattcaag tgggagcgtg tgatgaactt
     2041 cgaggacggc ggggtcgtga ccgtgaccca ggacagctcc ctccaggacg gcgagttcat
     2101 ctacaaggtg aagctgaggg gcacaaactt cccctctgac ggccccgtga tgcagaagaa
     2161 aacaatgggc tgggaggcca gctcagaaag aatgtaccct gaggacggag ccctgaaagg
     2221 cgaaatcaaa caaagactga agctgaagga tggaggccac tacgacgccg aagtcaagac
     2281 aacctacaag gccaaaaagc ctgtgcagct gcctggcgcc tacaacgtga atatcaagct
     2341 ggacatcacc tcccacaacg aggactacac catcgtggag cagtacgagc gggccgaggg
     2401 cagacacagc accggcggca tggacgagct gtacaagtga gaattctgca gatatccagc
     2461 acagtggcgg ccgctcgagt ctagagggcc cgtttaaacc cgctgatcag cctcgactgt
     2521 gccttctagt tgccagccat ctgttgtttg cccctccccc gtgccttcct tgaccctgga
     2581 aggtgccact cccactgtcc tttcctaata aaatgaggaa attgcatcgc attgtctgag
     2641 taggtgtcat tctattctgg ggggtggggt ggggcaggac agcaaggggg aggattggga
     2701 agacaatagc aggcatgctg gggatgcggt gggctctatg gcttctgagg cggaaagaac
     2761 cagctggggc tctagggggt atccccacgc gccctgtagc ggcgcattaa gcgcggcggg
     2821 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2881 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2941 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     3001 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     3061 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     3121 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     3181 aaatgagctg atttaacaaa aatttaacgc gaattaattc tgtggaatgt gtgtcagtta
     3241 gggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat gcatctcaat
     3301 tagtcagcaa ccaggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     3361 atgcatctca attagtcagc aaccatagtc ccgcccctaa ctccgcccat cccgccccta
     3421 actccgccca gttccgccca ttctccgccc catggctgac taattttttt tatttatgca
     3481 gaggccgagg ccgcctctgc ctctgagcta ttccagaagt agtgaggagg cttttttgga
     3541 ggcctaggct tttgcaaaaa gctcccggga gcttgtatat ccattttcgg atctgatcaa
     3601 gagacaggat gaggatcgtt tcgcatgatt gaacaagatg gattgcacgc aggttctccg
     3661 gccgcttggg tggagaggct attcggctat gactgggcac aacagacaat cggctgctct
     3721 gatgccgccg tgttccggct gtcagcgcag gggcgcccgg ttctttttgt caagaccgac
     3781 ctgtccggtg ccctgaatga actgcaggac gaggcagcgc ggctatcgtg gctggccacg
     3841 acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg aagcgggaag ggactggctg
     3901 ctattgggcg aagtgccggg gcaggatctc ctgtcatctc accttgctcc tgccgagaaa
     3961 gtatccatca tggctgatgc aatgcggcgg ctgcatacgc ttgatccggc tacctgccca
     4021 ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta ctcggatgga agccggtctt
     4081 gtcgatcagg atgatctgga cgaagagcat caggggctcg cgccagccga actgttcgcc
     4141 aggctcaagg cgcgcatgcc cgacggcgag gatctcgtcg tgacccatgg cgatgcctgc
     4201 ttgccgaata tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg
     4261 ggtgtggcgg accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt
     4321 ggcggcgaat gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag
     4381 cgcatcgcct tctatcgcct tcttgacgag ttcttctgag cgggactctg gggttcgaaa
     4441 tgaccgacca agcgacgccc aacctgccat cacgagattt cgattccacc gccgccttct
     4501 atgaaaggtt gggcttcgga atcgttttcc gggacgccgg ctggatgatc ctccagcgcg
     4561 gggatctcat gctggagttc ttcgcccacc ccaacttgtt tattgcagct tataatggtt
     4621 acaaataaag caatagcatc acaaatttca caaataaagc atttttttca ctgcattcta
     4681 gttgtggttt gtccaaactc atcaatgtat cttatcatgt ctgtataccg tcgacctcta
     4741 gctagagctt ggcgtaatca tggtcatagc tgtttcctgt gtgaaattgt tatccgctca
     4801 caattccaca caacatacga gccggaagca taaagtgtaa agcctggggt gcctaatgag
     4861 tgagctaact cacattaatt gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt
     4921 cgtgccagct gcattaatga atcggccaac gcgcggggag aggcggtttg cgtattgggc
     4981 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     5041 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     5101 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     5161 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     5221 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     5281 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     5341 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     5401 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     5461 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     5521 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5581 ggcctaacta cggctacact agaagaacag tatttggtat ctgcgctctg ctgaagccag
     5641 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5701 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5761 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5821 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5881 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5941 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     6001 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     6061 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     6121 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     6181 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     6241 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     6301 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     6361 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     6421 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     6481 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6541 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6601 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6661 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6721 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6781 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6841 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6901 gaaaagtgcc acctgacgtc
//
