LOCUS       sequence-308411-        4765 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Expresses jack bean (Canavalia ensiformis) asparaginyl endopeptidase
            1 in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4765)
  AUTHORS   Bernath-Levin K, Nelson C, Elliott AG, Jayasena AS, Millar AH, Craik
            DJ, Mylne JS
  TITLE     Peptide macrocyclization by a bifunctional endoprotease.
  JOURNAL   Chem Biol. 2015 May 21;22(5):571-82. doi: 
            10.1016/j.chembiol.2015.04.010. Epub 2015 May 7.
  PUBMED    25960260
REFERENCE   2  (bases 1 to 4765)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1016/j.chembiol.2015.04.010"; journalName: "Chem Biol"; date: 
            "2015-05-21- 21"; volume: "22"; issue: "5"; pages: "571-82"
COMMENT     Sequence Label: pQE30-CeAEP1
FEATURES             Location/Qualifiers
     source          1..4765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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)."
     RBS             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             127..144
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1512..1606
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1650..2309
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1716..1735)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2374..2460
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2507..2529)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2615..2755
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2670..2689
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2770..2787)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2941..3529)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3021..3040)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3700..4491,4492..4560))
                     /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     4323..4342
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4561..4665)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4733..4751
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 ctcgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaga
      121 ggatcgcatc accatcacca tcacggatcc gaagttggca cccgttgggc agttctggtt
      181 gcaggtagca atggttatgg taattatcgt catcaggccg atgtttgtca tgcatatcag
      241 ctgctgatta aaggtggtgt gaaagaagaa aacatcgtcg tctttatgta cgatgatatt
      301 gcctacaatg ccatgaatcc gcgtccgggt gttattatta atcatccgca gggtccggat
      361 gtttatgccg gtgttccgaa agattatacc ggtgaagatg ttacaccgga aaatctgtat
      421 gcagttattc tgggcgacaa aagcaaagtt aaaggtggta gcggcaaagt gattaatagc
      481 aatccggaag atcgcatctt catcttttat agcgatcatg gtggtccggg tgttctgggt
      541 atgccgaatg caccgtttgt ttatgccatg gattttatcg acgtgctgaa aaagaaacat
      601 gccagcggtg gctataaaga aatggtgatt tatatcgaag cctgtgaaag cggcagcatt
      661 tttgaaggta ttatgccgaa agacctgaac atttatgtta ccaccgcaag caatgcccaa
      721 gaaaatagct ttggcaccta ttgtccgggt atgaatccgc ctccgcctga agaatatgtt
      781 acctgtctgg gtgatctgta tagcgttagc tggatggaag atagcgaaac ccataatctg
      841 aaacgtgaaa ccgttcagca gcagtatcag agcgttcgta aacgtaccag caatagcaat
      901 agctatcgtt ttggtagcca cgttatgcag tatggtgata ccaatatcac cgcagaaaaa
      961 ctgtatctgt atcatggttt tgatccggca accgttaatt ttccgcctca taatggtaat
     1021 ctggaagcca aaatggaagt tgttaatcag cgtgatgccg aactgctgtt tatgtggcag
     1081 atgtatcagc gtagcaatca tcagccggaa aaaaagaccc atatcctgga acaaatcacc
     1141 gaaaccgtga aacatcgtaa tcatctggat ggtagcgttg aactgattgg tgttctgctg
     1201 tatggtccgg gtaaaagcag cagcgttctg catagcgttc gtgcaccggg tctgccgctg
     1261 gttgatgatt ggacctgtct gaaaagcatg gttcgtgttt ttgaaaccca ttgtggtagc
     1321 ctgacccagt atggtatgaa acatatgcgt gcctttggca atgtttgtaa tagcggtgtt
     1381 agcaaagcca gcatggaaga ggcatgtaaa gcagcatgtg gtggttatga tgcaggtctg
     1441 ctgtatccga gcaataccgg ttatagcgca taataagtcg acctgcagcc aagcttaatt
     1501 agctgagctt ggactcctgt tgatagatcc agtaatgacc tcagaactcc atctggattt
     1561 gttcagaacg ctcggttgcc gccgggcgtt ttttattggt gagaatccaa gctagcttgg
     1621 cgagattttc aggagctaag gaagctaaaa tggagaaaaa aatcactgga tataccaccg
     1681 ttgatatatc ccaatggcat cgtaaagaac attttgaggc atttcagtca gttgctcaat
     1741 gtacctataa ccagaccgtt cagctggata ttacggcctt tttaaagacc gtaaagaaaa
     1801 ataagcacaa gttttatccg gcctttattc acattcttgc ccgcctgatg aatgctcatc
     1861 cggaatttcg tatggcaatg aaagacggtg agctggtgat atgggatagt gttcaccctt
     1921 gttacaccgt tttccatgag caaactgaaa cgttttcatc gctctggagt gaataccacg
     1981 acgatttccg gcagtttcta cacatatatt cgcaagatgt ggcgtgttac ggtgaaaacc
     2041 tggcctattt ccctaaaggg tttattgaga atatgttttt cgtctcagcc aatccctggg
     2101 tgagtttcac cagttttgat ttaaacgtgg ccaatatgga caacttcttc gcccccgttt
     2161 tcaccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg gcgattcagg
     2221 ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa ttacaacagt
     2281 actgcgatga gtggcagggc ggggcgtaat ttttttaagg cagttattgg tgcccttaaa
     2341 cgcctggggt aatgactctc tagcttgagg catcaaataa aacgaaaggc tcagtcgaaa
     2401 gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag taggacaaat
     2461 ccgccctcta gagctgcctc gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc
     2521 agctcccgga gacggtcaca gcttgtctgt aagcggatgc cgggagcaga caagcccgtc
     2581 agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg
     2641 atagcggagt gtatactggc ttaactatgc ggcatcagag cagattgtac tgagagtgca
     2701 ccatatgcgg tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgctc
     2761 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     2821 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     2881 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     2941 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3001 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     3061 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     3121 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     3181 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     3241 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     3301 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     3361 taactacggc tacactagaa ggacagtatt tggtatctgc gctctgctga agccagttac
     3421 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     3481 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     3541 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     3601 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     3661 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     3721 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     3781 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     3841 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     3901 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     3961 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     4021 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     4081 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     4141 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     4201 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     4261 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     4321 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     4381 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     4441 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     4501 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     4561 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     4621 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     4681 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
     4741 tatcacgagg ccctttcgtc ttcac
//
