LOCUS       sequence-325547-        4765 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses Arabidopsis thaliana asparaginyl endopeptidase 2 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 Dec 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-AtAEP2
FEATURES             Location/Qualifiers
     source          1..4765
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..15,16..21,22..38,39..45)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    21..37
                     /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    53..69
                     /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             88..99
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             118..135
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     terminator      1503..1597
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1641..2300
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1707..1726)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2365..2451
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2498..2520)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2606..2746
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2661..2680
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2761..2778)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2932..3520)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3012..3031)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3691..4482,4483..4551))
                     /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     4314..4333
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4552..4656)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4724..4742
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcataaaaaa tttatttgct ttgtgagcgg ataacaatta taatagattc aattgtgagc
       61 ggataacaat ttcacacaga attcattaaa gaggagaaat taactatgag aggatcgcat
      121 caccatcacc atcacggatc cggtgttggc acccgttggg cagttctggt tgcaggtagc
      181 agcggttatg gtaattatcg tcatcaggca gatgtttgtc atgcatatca gattctgcgt
      241 aaaggtggtc tgaaagaaga aaacatcgtc gttctgatgt atgatgatat tgccaatcat
      301 ccgctgaatc cgcgtccggg tacactgatt aatcatccgg atggtgatga tgtttatgcc
      361 ggtgttccga aagattatac cggtagcagc gttaccgcag caaattttta tgcagttctg
      421 ctgggtgatc agaaagcagt taaaggtggt agcggtaaag ttattgcaag caaaccgaac
      481 gatcacatct ttgtttatta tgcagatcat ggtggtccgg gtgttctggg tatgccgaat
      541 acaccgcata tttatgcagc cgattttatc gaaaccctga aaaagaaaca tgccagcggc
      601 acctataaag aaatggttat ttatgtggaa gcctgtgaaa gcggcagcat ttttgaaggt
      661 attatgccga aagacctgaa catttatgtt accaccgcaa gcaatgcaca agaaagcagc
      721 tatggcacct attgtccggg tatgaatccg agccctccga gcgaatatat tacctgtctg
      781 ggtgatctgt atagcgttgc atggatggaa gatagcgaaa cccataacct gaaaaaagaa
      841 accatcaaac agcagtacca caccgttaaa atgcgtacca gcaactacaa tacctatagc
      901 ggtggtagcc acgttatgga atatggtaac aacagcatca aaagcgagaa actgtatctg
      961 tatcagggtt ttgatccggc aaccgttaat ctgccgctga atgaactgcc ggttaaaagc
     1021 aaaattggtg ttgttaatca gcgtgatgcc gatctgctgt ttctgtggca tatgtatcgt
     1081 accagcgaag atggtagccg taaaaaagat gataccctga aagaactgac cgaaaccacc
     1141 cgtcatcgta aacatctgga tgcaagcgtt gaactgattg caaccattct gtttggtccg
     1201 accatgaatg ttctgaatct ggttcgtgaa ccgggtctgc cgctggttga tgattgggaa
     1261 tgtctgaaaa gcatggttcg tgtgtttgaa gaacattgtg gtagcctgac ccagtatggt
     1321 atgaaacata tgcgtgcatt tgccaatgtg tgcaataatg gtgttagcaa agaactgatg
     1381 gaagaggcaa gcaccgcagc atgtggtggt tatagcgaag cacgttatac cgttcatccg
     1441 agcattctgg gttatagcgc ataataagtc gacctgcagc caagcttaat tagctgagct
     1501 tggactcctg ttgatagatc cagtaatgac ctcagaactc catctggatt tgttcagaac
     1561 gctcggttgc cgccgggcgt tttttattgg tgagaatcca agctagcttg gcgagatttt
     1621 caggagctaa ggaagctaaa atggagaaaa aaatcactgg atataccacc gttgatatat
     1681 cccaatggca tcgtaaagaa cattttgagg catttcagtc agttgctcaa tgtacctata
     1741 accagaccgt tcagctggat attacggcct ttttaaagac cgtaaagaaa aataagcaca
     1801 agttttatcc ggcctttatt cacattcttg cccgcctgat gaatgctcat ccggaatttc
     1861 gtatggcaat gaaagacggt gagctggtga tatgggatag tgttcaccct tgttacaccg
     1921 ttttccatga gcaaactgaa acgttttcat cgctctggag tgaataccac gacgatttcc
     1981 ggcagtttct acacatatat tcgcaagatg tggcgtgtta cggtgaaaac ctggcctatt
     2041 tccctaaagg gtttattgag aatatgtttt tcgtctcagc caatccctgg gtgagtttca
     2101 ccagttttga tttaaacgtg gccaatatgg acaacttctt cgcccccgtt ttcaccatgg
     2161 gcaaatatta tacgcaaggc gacaaggtgc tgatgccgct ggcgattcag gttcatcatg
     2221 ccgtttgtga tggcttccat gtcggcagaa tgcttaatga attacaacag tactgcgatg
     2281 agtggcaggg cggggcgtaa tttttttaag gcagttattg gtgcccttaa acgcctgggg
     2341 taatgactct ctagcttgag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc
     2401 tttcgtttta tctgttgttt gtcggtgaac gctctcctga gtaggacaaa tccgccctct
     2461 agagctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg
     2521 agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt
     2581 cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag
     2641 tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg
     2701 gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc
     2761 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     2821 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2881 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2941 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3001 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3061 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3121 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3181 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     3241 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3301 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3361 ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3421 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3481 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3541 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3601 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3661 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3721 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     3781 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     3841 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     3901 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     3961 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4021 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4081 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4141 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4201 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4261 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4321 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     4381 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4441 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4501 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     4561 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     4621 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     4681 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     4741 gccctttcgt cttcacctcg agaaa
//
