LOCUS       Exported                5678 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  expression plasmid for C-terminal his tagged S. aureus SrtA with 
            enhanced catalytic activity.
ACCESSION   .
VERSION     .
KEYWORDS    pet30b-5M SrtA
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5678)
  AUTHORS   Shi J, Kundrat L, Pishesha N, Bilate A, Theile C, Maruyama T, Dougan
            SK, Ploegh HL, Lodish HF
  TITLE     Engineered red blood cells as carriers for systemic delivery of a 
            wide array of functional probes.
  JOURNAL   Proc Natl Acad Sci U S A. 2014 Jul 15;111(28):10131-6. doi: 
            10.1073/pnas.1409861111. Epub 2014 Jun 30.
  PUBMED    24982154
REFERENCE   2  (bases 1 to 5678)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..5678
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             520..537
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(590..608)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      604..651
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      688..1143
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(775..794)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     985..1006
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1235..2050)
                     /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     1958..1977
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2042..2061
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2172..2760
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2661..2680
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2914..2931
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2946..3085
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3011..3030)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3171..3193
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3187..3378)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4187..5269)
                     /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     5231..5250
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5270..5347)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5553..5572
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5660..1
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5660..5678
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatatacata tgcaagctaa acctcaaatt ccgaaagata aatcaaaagt ggcaggctat
      121 attgaaattc cagatgctga tattaaagaa ccagtatatc caggaccagc aacacgcgaa
      181 caattaaata gaggtgtaag ctttgcagaa gaaaatgaat cactagatga tcaaaatatt
      241 tcaattgcag gacacacttt cattgaccgt ccgaactatc aatttacaaa tcttaaagca
      301 gccaaaaaag gtagtatggt gtactttaaa gttggtaatg aaacacgtaa gtataaaatg
      361 acaagtataa gaaacgttaa gccaacagct gtagaagttc tagatgaaca aaaaggtaaa
      421 gataaacaat taacattaat tacttgtgat gattacaatg aagagacagg cgtttgggaa
      481 acacgtaaaa tctttgtagc tacagaagtc aaactcgagc accaccacca ccaccactga
      541 gatccggctg ctaacaaagc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
      601 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
      661 ggaactatat ccggattggc gaatgggacg cgccctgtag cggcgcatta agcgcggcgg
      721 gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag cgccctagcg cccgctcctt
      781 tcgctttctt cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc
      841 gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
      901 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
      961 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca acactcaacc
     1021 ctatctcggt ctattctttt gatttataag ggattttgcc gatttcggcc tattggttaa
     1081 aaaatgagct gatttaacaa aaatttaacg cgaattttaa caaaatatta acgcttacaa
     1141 tttaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     1201 acattcaaat atgtatccgc tcatgaatta attcttagaa aaactcatcg agcatcaaat
     1261 gaaactgcaa tttattcata tcaggattat caataccata tttttgaaaa agccgtttct
     1321 gtaatgaagg agaaaactca ccgaggcagt tccataggat ggcaagatcc tggtatcggt
     1381 ctgcgattcc gactcgtcca acatcaatac aacctattaa tttcccctcg tcaaaaataa
     1441 ggttatcaag tgagaaatca ccatgagtga cgactgaatc cggtgagaat ggcaaaagtt
     1501 tatgcatttc tttccagact tgttcaacag gccagccatt acgctcgtca tcaaaatcac
     1561 tcgcatcaac caaaccgtta ttcattcgtg attgcgcctg agcgagacga aatacgcgat
     1621 cgctgttaaa aggacaatta caaacaggaa tcgaatgcaa ccggcgcagg aacactgcca
     1681 gcgcatcaac aatattttca cctgaatcag gatattcttc taatacctgg aatgctgttt
     1741 tcccggggat cgcagtggtg agtaaccatg catcatcagg agtacggata aaatgcttga
     1801 tggtcggaag aggcataaat tccgtcagcc agtttagtct gaccatctca tctgtaacat
     1861 cattggcaac gctacctttg ccatgtttca gaaacaactc tggcgcatcg ggcttcccat
     1921 acaatcgata gattgtcgca cctgattgcc cgacattatc gcgagcccat ttatacccat
     1981 ataaatcagc atccatgttg gaatttaatc gcggcctaga gcaagacgtt tcccgttgaa
     2041 tatggctcat aacacccctt gtattactgt ttatgtaagc agacagtttt attgttcatg
     2101 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     2161 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     2221 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     2281 gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta gccgtagtta
     2341 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     2401 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     2461 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     2521 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     2581 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     2641 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     2701 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     2761 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     2821 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     2881 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggaa
     2941 gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcaatggt
     3001 gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca ctccgctatc
     3061 gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg acgcgccctg
     3121 acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct ccgggagctg
     3181 catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc ggtaaagctc
     3241 atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt ccagctcgtt
     3301 gagtttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt taagggcggt
     3361 tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca tgggggtaat
     3421 gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg aacatgcccg
     3481 gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg accagagaaa
     3541 aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc cacagggtag
     3601 ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg acttccgcgt
     3661 ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc aggtcgcaga
     3721 cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat tctgctaacc
     3781 agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga tcatgcgcac
     3841 ccgtggggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt tggtggcggg
     3901 accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa gcgacaggcc
     3961 gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga gcgctgccgg
     4021 cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga cgatagtcat
     4081 gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga
     4141 tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt
     4201 ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg
     4261 cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg ggcaacagct
     4321 gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc
     4381 ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat gagctgtctt
     4441 cggtatcgtc gtatcccact accgagatgt ccgcaccaac gcgcagcccg gactcggtaa
     4501 tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca gtgggaacga
     4561 tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc cagtcgcctt
     4621 cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag ccagccagac
     4681 gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc tggtgaccca
     4741 atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa ataatactgt
     4801 tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg caggcagctt
     4861 ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca ctgacgcgtt
     4921 gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt tctaccatcg
     4981 acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg acaatttgcg
     5041 acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac tgtttgcccg
     5101 ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc gcttccactt
     5161 tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa acggtctgat
     5221 aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca ttcaccaccc
     5281 tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg cgccattcga
     5341 tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa gcagcccagt
     5401 agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg
     5461 cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca agcgctcatg
     5521 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     5581 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatcgagatc
     5641 gatctcgatc ccgcgaaatt aatacgactc actatagg
//
