LOCUS       sequence-173616-        5699 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses calcium-dependent Sortase A with a TEV cleavage site 
            proximal to the C-terminal histidine tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5699)
  AUTHORS   Sarpong K, Bose R
  TITLE     Efficient sortase-mediated N-terminal labeling of TEV protease 
            cleaved recombinant proteins.
  JOURNAL   Anal Biochem. 2017 Mar 15;521:55-58. doi: 10.1016/j.ab.2017.01.008. 
            Epub 2017 Jan 11.
  PUBMED    28088451
REFERENCE   2  (bases 1 to 5699)
  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: "Anal 
            Biochem."; date: "2017-03-15"; volume: "521:55-58. doi"; pages: " 
            10.1016/j.ab.2017.01.008. Epub 2017 Jan 11"
COMMENT     Sequence Label: pET30b-5M SrtA with TEV site
FEATURES             Location/Qualifiers
     source          1..5699
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(8..26)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      22..69
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      106..561
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(193..212)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     403..424
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(653..1468)
                     /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             complement(653..1468)
                     /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     1376..1395
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1460..1479
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      1590..2178
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2079..2098
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2332..2349
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2364..2503
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2429..2448)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2589..2611
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(2605..2796)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(3605..4687)
                     /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     4649..4668
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4688..4765)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     4971..4990
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5078..5097
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5078..5096
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5097..5121
                     /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             5136..5158
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5616..5636
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             5637..5654
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
ORIGIN
        1 gctgccaccg ctgagcaata actagcataa ccccttgggg cctctaaacg ggtcttgagg
       61 ggttttttgc tgaaaggagg aactatatcc ggattggcga atgggacgcg ccctgtagcg
      121 gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg
      181 ccctagcgcc cgctcctttc gctttcttcc cttcctttct cgccacgttc gccggctttc
      241 cccgtcaagc tctaaatcgg gggctccctt tagggttccg atttagtgct ttacggcacc
      301 tcgaccccaa aaaacttgat tagggtgatg gttcacgtag tgggccatcg ccctgataga
      361 cggtttttcg ccctttgacg ttggagtcca cgttctttaa tagtggactc ttgttccaaa
      421 ctggaacaac actcaaccct atctcggtct attcttttga tttataaggg attttgccga
      481 tttcggccta ttggttaaaa aatgagctga tttaacaaaa atttaacgcg aattttaaca
      541 aaatattaac gcttacaatt taggtggcac ttttcgggga aatgtgcgcg gaacccctat
      601 ttgtttattt ttctaaatac attcaaatat gtatccgctc atgaattaat tcttagaaaa
      661 actcatcgag catcaaatga aactgcaatt tattcatatc aggattatca ataccatatt
      721 tttgaaaaag ccgtttctgt aatgaaggag aaaactcacc gaggcagttc cataggatgg
      781 caagatcctg gtatcggtct gcgattccga ctcgtccaac atcaatacaa cctattaatt
      841 tcccctcgtc aaaaataagg ttatcaagtg agaaatcacc atgagtgacg actgaatccg
      901 gtgagaatgg caaaagttta tgcatttctt tccagacttg ttcaacaggc cagccattac
      961 gctcgtcatc aaaatcactc gcatcaacca aaccgttatt cattcgtgat tgcgcctgag
     1021 cgagacgaaa tacgcgatcg ctgttaaaag gacaattaca aacaggaatc gaatgcaacc
     1081 ggcgcaggaa cactgccagc gcatcaacaa tattttcacc tgaatcagga tattcttcta
     1141 atacctggaa tgctgttttc ccggggatcg cagtggtgag taaccatgca tcatcaggag
     1201 tacggataaa atgcttgatg gtcggaagag gcataaattc cgtcagccag tttagtctga
     1261 ccatctcatc tgtaacatca ttggcaacgc tacctttgcc atgtttcaga aacaactctg
     1321 gcgcatcggg cttcccatac aatcgataga ttgtcgcacc tgattgcccg acattatcgc
     1381 gagcccattt atacccatat aaatcagcat ccatgttgga atttaatcgc ggcctagagc
     1441 aagacgtttc ccgttgaata tggctcataa caccccttgt attactgttt atgtaagcag
     1501 acagttttat tgttcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     1561 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     1621 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     1681 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt
     1741 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     1801 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     1861 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     1921 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     1981 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     2041 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     2101 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     2161 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     2221 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     2281 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     2341 gtgagcgagg aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt
     2401 atttcacacc gcaatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc
     2461 agtatacact ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac
     2521 acccgctgac gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt
     2581 gaccgtctcc gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag
     2641 gcagctgcgg taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc
     2701 atccgcgtcc agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg
     2761 ggccatgtta agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt
     2821 tctgttcatg ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac
     2881 tgatgatgaa catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat
     2941 gcggcgggac cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt
     3001 aggtgttcca cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca
     3061 gggcgctgac ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt
     3121 tgttgctcag gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg
     3181 tgattcattc tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag
     3241 gagcacgatc atgcgcaccc gtggggccgc catgccggcg ataatggcct gcttctcgcc
     3301 gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca agattccgaa
     3361 taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct cgccgaaaat
     3421 gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga cagtcataag
     3481 tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt tgaaggctct
     3541 caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat taattgcgtt
     3601 gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg
     3661 ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca
     3721 gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc
     3781 ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga
     3841 tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatgtcc gcaccaacgc
     3901 gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca
     3961 gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca
     4021 tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt
     4081 tatgccagcc agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg
     4141 cgatttgctg gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat
     4201 gggagaaaat aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa
     4261 cattagtgca ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga
     4321 tcagcccact gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc
     4381 cgcttcgttc taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa
     4441 tcgccgcgac aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca
     4501 gcaacgactg tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg
     4561 ccatcgccgc ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca
     4621 cgcgggaaac ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg
     4681 gtttcacatt caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa
     4741 aggttttgcg ccattcgatg gtgtccggga tctcgacgct ctcccttatg cgactcctgc
     4801 attaggaagc agcccagtag taggttgagg ccgttgagca ccgccgccgc aaggaatggt
     4861 gcatgcaagg agatggcgcc caacagtccc ccggccacgg ggcctgccac catacccacg
     4921 ccgaaacaag cgctcatgag cccgaagtgg cgagcccgat cttccccatc ggtgatgtcg
     4981 gcgatatagg cgccagcaac cgcacctgtg gcgccggtga tgccggccac gatgcgtccg
     5041 gcgtagagga tcgagatcga tctcgatccc gcgaaattaa tacgactcac tataggggaa
     5101 ttgtgagcgg ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata
     5161 tacatatgca agctaaacct caaattccga aagataaatc aaaagtggca ggctatattg
     5221 aaattccaga tgctgatatt aaagaaccag tatatccagg accagcaaca cgcgaacaat
     5281 taaatagagg tgtaagcttt gcagaagaaa atgaatcact agatgatcaa aatatttcaa
     5341 ttgcaggaca cactttcatt gaccgtccga actatcaatt tacaaatctt aaagcagcca
     5401 aaaaaggtag tatggtgtac tttaaagttg gtaatgaaac acgtaagtat aaaatgacaa
     5461 gtataagaaa cgttaagcca acagctgtag aagttctaga tgaacaaaaa ggtaaagata
     5521 aacaattaac attaattact tgtgatgatt acaatgaaga gacaggcgtt tgggaaacac
     5581 gtaaaatctt tgtagctaca gaagtcaaac tcgaggagaa tctctatttt caaggtcacc
     5641 accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct gagttggct
//
