LOCUS       sequence-314615-        4837 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses sunflower (Helianthus annuus) asparaginyl endopeptidase 1 
            in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4837)
  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 4837)
  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-HaAEP1
FEATURES             Location/Qualifiers
     source          1..4837
                     /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      1584..1678
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1722..2381
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1788..1807)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2446..2532
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     complement(2579..2601)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    2687..2827
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     2742..2761
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(2842..2859)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3013..3601)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3093..3112)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3772..4563,4564..4632))
                     /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     4395..4414
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4633..4737)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4805..4823
                     /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 agcggtggtc agaaatggcg ttggggttgg
      181 gacccgctga ttcgtagtcc ggttgatgca gaacaagagg ttgatgagca gatgaccaat
      241 ggcaccaaat gggcagttct ggttgcaggt agcaaaggtt atggtaatta tcgtcatcag
      301 gccgatgttt gtcatgcata tcaggttctg aaaaaaggtg gcctgaaaga tgaaaacatc
      361 gtggtgttta tgtatgatga tatcgccaaa agcgaaatga atccgcgtcc gggtattatc
      421 attaatagcc cgaaaggtga agatgtttat gccggtgttc cgaaagatta taccggtaaa
      481 aatgtgaccg ttgataatct gagcgcagtt ctgctgggtg atcgtagcgc agttaaaggt
      541 ggtagcggta aagttgttga tagcaaaccg gaagatcgca tctttctgtt ttatagcgat
      601 catggtggtc cgggtgttct gggtatgccg aatgaaccgc atctggttgc caaagatctg
      661 gttgatgtgc tgaaaaaaaa acatgcaatg ggcacctaca aagaaatggt gatttatctg
      721 gaagcatgtg aaagcggcag catttttgaa ggtattctgc ctgaagatct gaacatttat
      781 gcaaccaccg caagcggtgc acaagaaaat agctatggca cctattgtcc gggtacagaa
      841 ccgagccctc cgcctgaata tatcacctgt ctgggcgatc tgtatagcgt tgcatggatg
      901 gaagatagcg aaacccataa cctgaaaaaa gaaagcctgg aacagcagtt taacaaagtg
      961 aaaaaacgta ccagcaacag caacacctat aataccggta gccatgttat ggaatatggc
     1021 agcaaagata ttaaaccgga aaaagtgtat ctgtatctgg gttttgatcc ggcaaccgtt
     1081 aatctgcctg caaatcagat tcattttgat aaactggatg gcgtgaatca gcgtgatgca
     1141 gatctgattt ttctgtggca gcgttacaaa aaaagcagcg aaagcacccg tccggaaatt
     1201 ctgcgtgaaa ttaccgaaac cctgacccat cgtggccatc tggatagcag cattgatatg
     1261 attggtgttc tgctgtttgg tccgcagaat ggtcgtagca ccctgcatag cgcacgtgca
     1321 ccgggtctgc ctctggtgga tgattgggaa tgttttaaaa gcaccgcacg cctgtttgaa
     1381 aaacattgtg gtctgctgac ccagtatggt atgaaacata tgcgtgcatt tgccaacatt
     1441 tgcaatagca gcgttgagaa aagcaaagtg gaagaagttt ttattgcaac ctgcggtggc
     1501 aaaaatatcg gtccgtatgg tacttttggt gcctatagcg tttaataagt cgacctgcag
     1561 ccaagcttaa ttagctgagc ttggactcct gttgatagat ccagtaatga cctcagaact
     1621 ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg gtgagaatcc
     1681 aagctagctt ggcgagattt tcaggagcta aggaagctaa aatggagaaa aaaatcactg
     1741 gatataccac cgttgatata tcccaatggc atcgtaaaga acattttgag gcatttcagt
     1801 cagttgctca atgtacctat aaccagaccg ttcagctgga tattacggcc tttttaaaga
     1861 ccgtaaagaa aaataagcac aagttttatc cggcctttat tcacattctt gcccgcctga
     1921 tgaatgctca tccggaattt cgtatggcaa tgaaagacgg tgagctggtg atatgggata
     1981 gtgttcaccc ttgttacacc gttttccatg agcaaactga aacgttttca tcgctctgga
     2041 gtgaatacca cgacgatttc cggcagtttc tacacatata ttcgcaagat gtggcgtgtt
     2101 acggtgaaaa cctggcctat ttccctaaag ggtttattga gaatatgttt ttcgtctcag
     2161 ccaatccctg ggtgagtttc accagttttg atttaaacgt ggccaatatg gacaacttct
     2221 tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg ctgatgccgc
     2281 tggcgattca ggttcatcat gccgtttgtg atggcttcca tgtcggcaga atgcttaatg
     2341 aattacaaca gtactgcgat gagtggcagg gcggggcgta atttttttaa ggcagttatt
     2401 ggtgccctta aacgcctggg gtaatgactc tctagcttga ggcatcaaat aaaacgaaag
     2461 gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa cgctctcctg
     2521 agtaggacaa atccgccctc tagagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc
     2581 tctgacacat gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca
     2641 gacaagcccg tcagggcgcg tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc
     2701 agtcacgtag cgatagcgga gtgtatactg gcttaactat gcggcatcag agcagattgt
     2761 actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg
     2821 catcaggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg
     2881 gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa
     2941 cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc
     3001 gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc
     3061 aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag
     3121 ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct
     3181 cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta
     3241 ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc
     3301 cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc
     3361 agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt
     3421 gaagtggtgg cctaactacg gctacactag aaggacagta tttggtatct gcgctctgct
     3481 gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc
     3541 tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca
     3601 agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta
     3661 agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     3721 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     3781 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     3841 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     3901 aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc
     3961 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     4021 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     4081 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     4141 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     4201 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     4261 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     4321 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     4381 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     4441 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     4501 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     4561 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     4621 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     4681 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     4741 atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta
     4801 taaaaatagg cgtatcacga ggccctttcg tcttcac
//
