LOCUS       sequence-422741-        6528 bp    DNA     circular SYN 14-APR-2025
DEFINITION  Expresses SpyTag-SpyCatcher mediated cyclized version of TEM-1 
            beta-lactamase (BLA). Cyclization of BLA led to an increase in 
            thermal resilience of >60 degrees celsius..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6528)
  AUTHORS   Schoene C, Fierer JO, Bennett SP, Howarth M
  TITLE     SpyTag/SpyCatcher cyclization confers resilience to boiling on a 
            mesophilic enzyme.
  JOURNAL   Angew Chem Int Ed Engl. 2014 Jun 10;53(24):6101-4. doi: 
            10.1002/anie.201402519. Epub 2014 May 9.
  PUBMED    24817566
REFERENCE   2  (bases 1 to 6528)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 14, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; doi: 
            "10.1002/anie.201402519"; journalName: "Angew Chem Int Ed Engl"; 
            date: "2014-06-10- 10"; volume: "53"; issue: "24"; pages: "6101-4"
COMMENT     Sequence Label: SpyTag-beta-Lactamase-SpyCatcher
FEATURES             Location/Qualifiers
     source          1..6528
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(14..61)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     57..75
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(128..145)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(539..1324)
                     /codon_start=1
                     /gene="bla(M)"
                     /product="beta-lactamase lacking the signal sequence"
                     /label=bla(M)
                     /note="allows cytosolic expression of beta-lactamase"
                     /translation="PETLVKVKDAEDQLGARVGYIELDLNSGKILESFRPEERFPMMST
                     FKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYSPVTEKHLTDGMTVRELCSAAITMS
                     DNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRWEPELNEAIPNDERDTTMPAAMATT
                     LRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSALPAGWFIADKSGAGERGSRGIIAA
                     LGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGASLIKHW"
     primer_bind     1159..1178
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     CDS             complement(1394..1411)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(1421..1438)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             1457..1479
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    1494..1518
                     /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)."
     primer_bind     complement(1518..1537)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1519..1537)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1621..1640)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        1846..1923
                     /gene="lacI"
                     /label=lacI promoter
     CDS             1924..3006
                     /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     complement(1943..1962)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             3815..4006
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(4000..4022)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4108..4247
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4163..4182
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4262..4279)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4433..5021)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4513..4532)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(5132..5151)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             5143..5958
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             5143..5958
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(5216..5235)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(6050..6505)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6187..6208)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6399..6418
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta
       61 gttattgctc agcggtggca gcagccaact cagcttcctt tcgggctttg ttagcagccg
      121 gatctcagtg gtggtggtgg tggtgctcga gtgcggccgc aagctttcat taaatatgag
      181 cgtcaccttt agttgctttg ccatttacag taacctgacc ttgctcatta actgtaaagg
      241 taatagcagt tgctacctca taaccgtctg gtgctgcggt ttcgacaaat gtatattttc
      301 ctggatacag gtagaaatct ttcacttgtc catctgaaat ccatgtacta atagttttac
      361 cagatgaatc acgcaactcc atagttgcac cagctaactc tttgccgtcc tcatcacgtt
      421 ttgagaattt aatatgggta gcactatctt cttcaattgt catatcaccg gactgacctt
      481 gctcacttga taaacctgat aaggtatcaa ccatggcgcc tccgctgcca ccactccccc
      541 aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg
      601 cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
      661 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
      721 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc atccagtcta
      781 ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg cgcaacgttg
      841 ttgccattgc tgcaggcatc gtggtgtcac gctcgtcgtt tggtatggct tcattcagct
      901 ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta
      961 gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
     1021 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
     1081 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg agttgctctt
     1141 gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa gtgctcatca
     1201 ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg agatccagtt
     1261 cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt
     1321 ctgggtgacc ggaaccccct gaacccttcg tcggcttgta ggcgtccacc atcacgatgt
     1381 gggctcccat atggctgccg cgcggcacca ggccgctgct gtgatgatga tgatgatggc
     1441 tgctgcccat ggtatatctc cttcttaaag ttaaacaaaa ttatttctag aggggaattg
     1501 ttatccgctc acaattcccc tatagtgagt cgtattaatt tcgcgggatc gagatctcga
     1561 tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg gttgctggcg
     1621 cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg ctcatgagcg
     1681 cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg ggcgccatct
     1741 ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta ctactgggct
     1801 gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac catcgaatgg
     1861 cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt cagggtggtg
     1921 aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc ttatcagacc
     1981 gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga aaaagtggaa
     2041 gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact ggcgggcaaa
     2101 cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc gtcgcaaatt
     2161 gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt gtcgatggta
     2221 gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc gcaacgcgtc
     2281 agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt ggaagctgcc
     2341 tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat caacagtatt
     2401 attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc attgggtcac
     2461 cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct gcgtctggct
     2521 ggctggcata aatatctcac tcgcaatcaa attcagccga tagcggaacg ggaaggcgac
     2581 tggagtgcca tgtccggttt tcaacaaacc atgcaaatgc tgaatgaggg catcgttccc
     2641 actgcgatgc tggttgccaa cgatcagatg gcgctgggcg caatgcgcgc cattaccgag
     2701 tccgggctgc gcgttggtgc ggatatctcg gtagtgggat acgacgatac cgaagacagc
     2761 tcatgttata tcccgccgtt aaccaccatc aaacaggatt ttcgcctgct ggggcaaacc
     2821 agcgtggacc gcttgctgca actctctcag ggccaggcgg tgaagggcaa tcagctgttg
     2881 cccgtctcac tggtgaaaag aaaaaccacc ctggcgccca atacgcaaac cgcctctccc
     2941 cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg
     3001 cagtgagcgc aacgcaatta atgtaagtta gctcactcat taggcaccgg gatctcgacc
     3061 gatgcccttg agagccttca acccagtcag ctccttccgg tgggcgcggg gcatgactat
     3121 cgtcgccgca cttatgactg tcttctttat catgcaactc gtaggacagg tgccggcagc
     3181 gctctgggtc attttcggcg aggaccgctt tcgctggagc gcgacgatga tcggcctgtc
     3241 gcttgcggta ttcggaatct tgcacgccct cgctcaagcc ttcgtcactg gtcccgccac
     3301 caaacgtttc ggcgagaagc aggccattat cgccggcatg gcggccccac gggtgcgcat
     3361 gatcgtgctc ctgtcgttga ggacccggct aggctggcgg ggttgcctta ctggttagca
     3421 gaatgaatca ccgatacgcg agcgaacgtg aagcgactgc tgctgcaaaa cgtctgcgac
     3481 ctgagcaaca acatgaatgg tcttcggttt ccgtgtttcg taaagtctgg aaacgcggaa
     3541 gtcagcgccc tgcaccatta tgttccggat ctgcatcgca ggatgctgct ggctaccctg
     3601 tggaacacct acatctgtat taacgaagcg ctggcattga ccctgagtga tttttctctg
     3661 gtcccgccgc atccataccg ccagttgttt accctcacaa cgttccagta accgggcatg
     3721 ttcatcatca gtaacccgta tcgtgagcat cctctctcgt ttcatcggta tcattacccc
     3781 catgaacaga aatccccctt acacggaggc atcagtgacc aaacaggaaa aaaccgccct
     3841 taacatggcc cgctttatca gaagccagac attaacgctt ctggagaaac tcaacgagct
     3901 ggacgcggat gaacaggcag acatctgtga atcgcttcac gaccacgctg atgagcttta
     3961 ccgcagctgc ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc
     4021 ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc
     4081 gtcagcgggt gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg
     4141 agtgtatact ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccattgc
     4201 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     4261 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4321 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4381 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4441 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4501 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4561 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4621 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4681 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4741 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     4801 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     4861 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     4921 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     4981 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5041 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgaaca
     5101 ataaaactgt ctgcttacat aaacagtaat acaaggggtg ttatgagcca tattcaacgg
     5161 gaaacgtctt gctctaggcc gcgattaaat tccaacatgg atgctgattt atatgggtat
     5221 aaatgggctc gcgataatgt cgggcaatca ggtgcgacaa tctatcgatt gtatgggaag
     5281 cccgatgcgc cagagttgtt tctgaaacat ggcaaaggta gcgttgccaa tgatgttaca
     5341 gatgagatgg tcagactaaa ctggctgacg gaatttatgc ctcttccgac catcaagcat
     5401 tttatccgta ctcctgatga tgcatggtta ctcaccactg cgatccccgg gaaaacagca
     5461 ttccaggtat tagaagaata tcctgattca ggtgaaaata ttgttgatgc gctggcagtg
     5521 ttcctgcgcc ggttgcattc gattcctgtt tgtaattgtc cttttaacag cgatcgcgta
     5581 tttcgtctcg ctcaggcgca atcacgaatg aataacggtt tggttgatgc gagtgatttt
     5641 gatgacgagc gtaatggctg gcctgttgaa caagtctgga aagaaatgca taaacttttg
     5701 ccattctcac cggattcagt cgtcactcat ggtgatttct cacttgataa ccttattttt
     5761 gacgagggga aattaatagg ttgtattgat gttggacgag tcggaatcgc agaccgatac
     5821 caggatcttg ccatcctatg gaactgcctc ggtgagtttt ctccttcatt acagaaacgg
     5881 ctttttcaaa aatatggtat tgataatcct gatatgaata aattgcagtt tcatttgatg
     5941 ctcgatgagt ttttctaaga attaattcat gagcggatac atatttgaat gtatttagaa
     6001 aaataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccaccta aattgtaagc
     6061 gttaatattt tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa
     6121 taggccgaaa tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt
     6181 gttgttccag tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg
     6241 cgaaaaaccg tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt
     6301 ttggggtcga ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga
     6361 gcttgacggg gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg
     6421 ggcgctaggg cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg
     6481 cttaatgcgc cgctacaggg cgcgtcccat tcgccaatcc ggatatag
//
