LOCUS       sequence-444819-        4958 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Mammalian expression vector encoding diptheria toxin A.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4958)
  AUTHORS   T. D. Luong, N. Martel, J. Rae, H. P. Lo, Y-W. Lim, Y. Wu, K-A. 
            McMahon, N. Fletcher, K. Thurecht, A.P.R. Johnston, N. Ariotti, T. 
            E. Hall, R.G. Parton
  TITLE     A modular encapsulation system for precision delivery of proteins, 
            nucleic acids and therapeutics
  JOURNAL   bioRxiv
REFERENCE   2  (bases 1 to 4958)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv"
COMMENT     Sequence Label: DTA in pCDEST2
FEATURES             Location/Qualifiers
     source          1..4958
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..53
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     35..52
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     protein_bind    108..132
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      136..145
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             151..723
                     /codon_start=1
                     /gene="Corynebacterium diphtheriae tox"
                     /product="diphtheria toxin fragment A"
                     /label=DTA
                     /translation="DDVVDSSKSFVMENFSSYHGTKPGYVDSIQKGIQKPKSGTQGNYD
                     DDWKGFYSTDNKYDAAGYSVDNENPLSGKAGGVVKVTYPGLTKVLALKVDNAETIKKEL
                     GLSLTEPLMEQVGTEEFIKRFGDGASRVVLSLPFAEGSSSVEYINNWEQAKALSVELEI
                     NFETRGKRGQDAMYEYMAQACAGNRVRR"
     protein_bind    complement(727..751)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     polyA_signal    753..887
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     protein_bind    complement(950..970)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     polyA_signal    1010..1144
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1257..1277)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(1257..1275)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(1296..1312)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1296..1312)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1309..1331)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    1320..1336
                     /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(1344..1350,1351..1368,1369..1374))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    1389..1410
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(1527..1544)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1698..2286)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1778..1797)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(2457..3248,3249..3317))
                     /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     3080..3099
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(3318..3422)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(3448..3903)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3585..3606)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3797..3816
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3974..4958
                     /label=CMV IE94 promoter
                     /note="enhancer/promoter region of simian cytomegalovirus 
                     major immediate early transcription unit IE94"
ORIGIN
        1 cgccattctg cctggggacg tcggagcaag cttgatttag gtgacactat agaatacaag
       61 ctacttgttc tttttgcagg atcccatcga ttcgaattca aggatcaaca agtttgtaca
      121 aaaaagcagg ctgccgccac catgggcgct gatgatgttg ttgattcttc taaatctttt
      181 gtgatggaaa acttttcttc gtaccacggg actaaacctg gttatgtaga ttccattcaa
      241 aaaggtatac aaaagccaaa atctggtaca caaggaaatt atgacgatga ttggaaaggg
      301 ttttatagta ccgacaataa atacgacgct gcgggatact ctgtagataa tgaaaacccg
      361 ctctctggaa aagctggagg cgtggtcaaa gtgacgtatc caggactgac gaaggttctc
      421 gcactaaaag tggataatgc cgaaactatt aagaaagagt taggtttaag tctcactgaa
      481 ccgttgatgg agcaagtcgg aacggaagag tttatcaaaa ggttcggtga tggtgcttcg
      541 cgtgtagtgc tcagccttcc cttcgctgag gggagttcta gcgttgaata tattaataac
      601 tgggaacagg cgaaagcgtt aagcgtagaa cttgagatta attttgaaac ccgtggaaaa
      661 cgtggccaag atgcgatgta tgagtatatg gctcaagcct gtgcaggaaa tcgtgtcagg
      721 cgataaaccc agctttcttg tacaaagtgg gggatccaga catgataaga tacattgatg
      781 agtttggaca aaccacaact agaatgcagt gaaaaaaatg ctttatttgt gaaatttgtg
      841 atgctattgc tttatttgta accattataa gctgcaataa acaagttaac aacaacaatt
      901 gcattcattt tatgtttcag gttcaggggg aggtgtggga ggttttttcc aactttatta
      961 tacatagttg cactcgagcc tctagaacta tagtgagtcg tattacgtag atccagacat
     1021 gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa aaaaatgctt
     1081 tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct gcaataaaca
     1141 agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg tgtgggaggt
     1201 tttttaattc gcggccgcgg cgccaatgca ttgggcccgg tacccagctt ttgttccctt
     1261 tagtgagggt taattgcgcg cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat
     1321 tgttatccgc tcacaattcc acacaacata cgagccggga gcataaagtg taaagcctgg
     1381 ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag
     1441 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     1501 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg
     1561 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg
     1621 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
     1681 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga
     1741 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct
     1801 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc
     1861 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg
     1921 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc
     1981 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
     2041 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag
     2101 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct
     2161 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc
     2221 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga
     2281 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca
     2341 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
     2401 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac
     2461 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt
     2521 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt
     2581 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag
     2641 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct
     2701 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
     2761 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc
     2821 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt
     2881 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg
     2941 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg
     3001 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct
     3061 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
     3121 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt
     3181 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt
     3241 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg
     3301 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat
     3361 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg
     3421 cgcacatttc cccgaaaagt gccacctaaa ttgtaagcgt taatattttg ttaaaattcg
     3481 cgttaaattt ttgttaaatc agctcatttt ttaaccaata ggccgaaatc ggcaaaatcc
     3541 cttataaatc aaaagaatag accgagatag ggttgagtgt tgttccagtt tggaacaaga
     3601 gtccactatt aaagaacgtg gactccaacg tcaaagggcg aaaaaccgtc tatcagggcg
     3661 atggcccact acgtgaacca tcaccctaat caagtttttt ggggtcgagg tgccgtaaag
     3721 cactaaatcg gaaccctaaa gggagccccc gatttagagc ttgacgggga aagccggcga
     3781 acgtggcgag aaaggaaggg aagaaagcga aaggagcggg cgctagggcg ctggcaagtg
     3841 tagcggtcac gctgcgcgta accaccacac ccgccgcgct taatgcgccg ctacagggcg
     3901 cgtcccattc gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt
     3961 cgctattacg ccagtcgacc gccaattcaa tatggcgtat atggactcat gccaattcaa
     4021 tatggtggat ctggacctgt gccaattcaa tatggcgtat atggactcgt gccaattcaa
     4081 tatggtggat ctggacccca gccaattcaa tatggcggac ttggcaccat gccaattcaa
     4141 tatggcggac ctggcactgt gccaactggg gaggggtcta cttggcacgg tgccaagttt
     4201 gaggaggggt cttggccctg tgccaagtcc gccatattga attggcatgg tgccaataat
     4261 ggcggccata ttggctatat gccaggatca atatataggc aatatccaat atggccctat
     4321 gccaatatgg ctattggcca ggttcaatac tatgtattgg ccctatgcca tatagtattc
     4381 catatatggg ttttcctatt gacgtagata gcccctccca atgggcggtc ccatatacca
     4441 tatatggggc ttcctaatac cgcccatagc cactccccca ttgacgtcaa tggtctctat
     4501 atatggtctt tcctattgac gtcatatggg cggtcctatt gacgtatatg gcgcctcccc
     4561 cattgacgtc aattacggta aatggcccgc ctggctcaat gcccattgac gtcaatagga
     4621 ccacccacca ttgacgtcaa tgggatggct cattgcccat tcatatccgt tctcacgccc
     4681 cctattgacg tcaatgacgg taaatggccc acttggcagt acatcaatat ctattaatag
     4741 taacttggca agtacattac tattggaagt acgccagggt acattggcag tactcccatt
     4801 gacgtcaatg gcggtaaatg gcccgcgatg gctgccaagt acatccccat tgacgtcaat
     4861 ggggaggggc aatgacgcaa atgggcgttc cattgacgta aatgggcggt aggcgtgcct
     4921 aatgggaggt ctatataagc aatgctcgtt tagggaac
//
