LOCUS       sequence-331113-        5828 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Cleavage of double glycine motif in peptides to remove the leader 
            sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5828)
  AUTHORS   Bobeica SC, Dong SH, Huo L, Mazo N, McLaughlin MI, Jimenez-Oses G, 
            Nair SK, van der Donk WA
  TITLE     Insights into AMS/PCAT transporters from biochemical and structural 
            characterization of a double Glycine motif protease.
  JOURNAL   Elife. 2019 Jan 14;8. pii: 42305. doi: 10.7554/eLife.42305.
  PUBMED    30638446
REFERENCE   2  (bases 1 to 5828)
  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: "Elife."; 
            date: "2019-01-14"; volume: "8. pii: 42305. doi"; pages: " 
            10.7554/eLife.42305"
COMMENT     Sequence Label: pETDuet-LahT150
FEATURES             Location/Qualifiers
     source          1..5828
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        24..42
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    43..67
                     /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             82..104
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             123..140
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     promoter        665..683
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    684..708
                     /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)."
     CDS             817..861
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     primer_bind     complement(899..917)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      913..960
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      997..1452
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1084..1103)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1294..1315
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(1567..2358,2359..2427))
                     /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     2190..2209
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2428..2520)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      2601..3189
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3090..3109
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3343..3360
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3375..3514
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3440..3459)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3600..3622
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3616..3807)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4379..5461)
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     5423..5442
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5462..5539)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5745..5764
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gatcgatctc gatcccgcga aattaatacg actcactata ggggaattgt gagcggataa
       61 caattcccct ctagaaataa ttttgtttaa ctttaagaag gagatatacc atgggcagca
      121 gccatcacca tcatcaccac agccaggatc cgagtaaaaa gcagatacag cctgtcacaa
      181 gagggcgagc caaggttcct gttatcatgc agatggaagc tctcgaatgt ggcgcggcat
      241 cacttgcaat ggtgcttgct tattataaaa aatgggtacc gctcgagcag gtcagagttg
      301 actgcggtgt atctcgtgac ggttccaatg cactgaatgt gcttaaggca gcacgaaact
      361 acggtcttga agcgaagggt tatcgttatg aaccggagaa gctgaagaag gaaggcacct
      421 tcccgtgtat tatacactgg aattttaatc attttgttgt tcttaaaggt ttcaagggga
      481 aatatgcata tataaacgat cctgcaaagg gtgatgttaa aataccgatg gaggagtttg
      541 atcgttcttt tacgggtata tgtctcattt tcaagcctac tgatagattt gaacagtaag
      601 cggccgcata atgcttaagt cgaacagaaa gtaatcgtat tgtacacggc cgcataatcg
      661 aaattaatac gactcactat aggggaattg tgagcggata acaattcccc atcttagtat
      721 attagttaag tataagaagg agatatacat atggcagatc tcaattggat atcggccggc
      781 cacgcgatcg ctgacgtcgg taccctcgag tctggtaaag aaaccgctgc tgcgaaattt
      841 gaacgccagc acatggactc gtctactagc gcagcttaat taacctaggc tgctgccacc
      901 gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg
      961 ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa
     1021 gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
     1081 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
     1141 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca
     1201 aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc
     1261 gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa
     1321 cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct
     1381 attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
     1441 cgtttacaat ttctggcggc acgatggcat gagattatca aaaaggatct tcacctagat
     1501 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     1561 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     1621 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     1681 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     1741 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     1801 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     1861 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     1921 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     1981 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     2041 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     2101 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     2161 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     2221 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     2281 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     2341 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     2401 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatcatgat tgaagcattt
     2461 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     2521 taggtcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta
     2581 gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa
     2641 acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt
     2701 tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag
     2761 ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta
     2821 atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca
     2881 agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag
     2941 cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa
     3001 agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga
     3061 acaggagagc gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc
     3121 gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc
     3181 ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt
     3241 gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt
     3301 gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag
     3361 gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac
     3421 cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac
     3481 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga
     3541 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc
     3601 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg
     3661 gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc
     3721 cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt
     3781 aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat
     3841 gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga
     3901 acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga
     3961 ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc
     4021 acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga
     4081 cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca
     4141 ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt
     4201 ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat
     4261 catgctagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     4321 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     4381 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4441 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     4501 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     4561 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     4621 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat gtccgcacca acgcgcagcc
     4681 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     4741 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     4801 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     4861 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     4921 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     4981 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     5041 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     5101 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     5161 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     5221 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     5281 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     5341 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     5401 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     5461 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     5521 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     5581 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     5641 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     5701 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     5761 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     5821 aggatcga
//
