LOCUS SpyCatcher003 S49C 4076 bp ds-DNA circular SYN 12-MAY-2021
DEFINITION Expresses SpyCatcher003 S49C in bacterial cytoplasm, with a unique
cysteine for chemical coupling.
ACCESSION .
VERSION .
KEYWORDS SpyCatcher003 S49C
SOURCE synthetic DNA construct
ORGANISM synthetic DNA construct
REFERENCE 1 (bases 1 to 4076)
AUTHORS Keeble AH, Turkki P, Stokes S, Khairil Anuar INA, Rahikainen R,
Hytonen VP, Howarth M
TITLE Approaching infinite affinity through engineering of peptide-protein
interaction.
JOURNAL Proc Natl Acad Sci U S A. 2019 Dec 10. pii: 1909653116. doi:
10.1073/pnas.1909653116.
PUBMED 31822621
REFERENCE 2 (bases 1 to 4076)
AUTHORS .
TITLE Direct Submission
JOURNAL Exported May 12, 2021 from SnapGene Server 1.1.58
//www.snapgene.com
FEATURES Location/Qualifiers
source 1..4076
/organism="synthetic DNA construct"
/mol_type="other DNA"
primer_bind 1..20
/label=T7
/note="T7 promoter, forward primer"
promoter 1..19
/label=T7 promoter
/note="promoter for bacteriophage T7 RNA polymerase"
RBS 51..73
/note="efficient ribosome binding site from bacteriophage
T7 gene 10 (Olins and Rangwala, 1989)"
CDS 93..110
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
CDS 132..152
/codon_start=1
/product="tobacco etch virus (TEV) protease recognition and
cleavage site"
/label=TEV site
/translation="ENLYFQG"
CDS 577..594
/codon_start=1
/product="6xHis affinity tag"
/label=6xHis
/translation="HHHHHH"
primer_bind complement(647..665)
/label=T7 Term
/note="T7 terminator, reverse primer"
terminator 661..708
/label=T7 terminator
/note="transcription terminator for bacteriophage T7 RNA
polymerase"
rep_origin 745..1200
/direction=RIGHT
/label=f1 ori
/note="f1 bacteriophage origin of replication; arrow
indicates direction of (+) strand synthesis"
primer_bind complement(832..851)
/label=F1ori-R
/note="F1 origin, reverse primer"
primer_bind 1042..1063
/label=F1ori-F
/note="F1 origin, forward primer"
promoter 1226..1330
/gene="bla"
/label=AmpR promoter
CDS 1331..2191
/codon_start=1
/gene="bla"
/product="beta-lactamase"
/label=AmpR
/note="confers resistance to ampicillin, carbenicillin, and
related antibiotics"
/translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
LIKHW"
primer_bind complement(1549..1568)
/label=Amp-R
/note="Ampicillin resistance gene, reverse primer"
rep_origin 2362..2950
/direction=RIGHT
/label=ori
/note="high-copy-number ColE1/pMB1/pBR322/pUC origin of
replication"
primer_bind 2851..2870
/label=pBR322ori-F
/note="pBR322 origin, forward primer"
primer_bind 3104..3121
/label=L4440
/note="L4440 vector, forward primer"
misc_feature 3136..3275
/label=bom
/note="basis of mobility region from pBR322"
primer_bind complement(3201..3220)
/label=pRS-marker
/note="pRS vectors, use to sequence yeast selectable
marker"
primer_bind 3361..3383
/label=pGEX 3'
/note="pGEX vectors, reverse primer"
CDS complement(3377..3568)
/codon_start=1
/gene="rop"
/product="Rop protein, which maintains plasmids at low copy
number"
/label=rop
/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
DELYRSCLARFGDDGENL"
ORIGIN
1 taatacgact cactataggg agaccacaac ggtttccctc tagaaataat tttgtttaac
61 tttaagaagg agatatacat atgtcgtact accatcacca tcaccatcac gattacgaca
121 tcccaacgac cgaaaacctg tattttcagg gcgccatggt aaccacctta tcaggtttat
181 caggtgagca aggtccgtcc ggtgatatga caactgaaga agatagtgct acccatatta
241 aattctcaaa acgtgatgag gacggccgtg agttagctgg tgcaactatg gagttgcgtg
301 attgctctgg taaaactatt agtacatgga tttcagatgg acatgtgaag gatttctacc
361 tgtatccagg aaaatataca tttgtcgaaa ccgcagcacc agacggttat gaggtagcaa
421 ctccaattga atttacagtt aatgaggacg gtcaggttac tgtagatggt gaagcaactg
481 aaggtgacgc tcatactgga tccagtggta gctaaatggt tgatgcttga ggatccgaat
541 tcgagctccg tcgacaagct tgcggccgca ctcgagcacc accaccacca ccactgagat
601 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
661 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
721 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
781 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
841 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
901 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
961 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
1021 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
1081 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
1141 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
1201 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
1261 ttcaaatatg tatccgctca tgagacaata accctggtaa atgcttcaat aatattgaaa
1321 aaggaagagt atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt
1381 ttgccttcct gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca
1441 gttgggtgca cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag
1501 ttttcgcccc gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc
1561 ggtattatcc cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca
1621 gaatgacttg gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt
1681 aagagaatta tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct
1741 gacaacgatc ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt
1801 aactcgcctt gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga
1861 caccacgatg cctgcagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact
1921 tactctagct tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc
1981 acttctgcgc tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga
2041 gcgtgggtct cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt
2101 agttatctac acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga
2161 gataggtgcc tcactgatta agcattggta actgtcagac caagtttact catatatact
2221 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
2281 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
2341 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
2401 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
2461 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgtcc ttctagtgta
2521 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
2581 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
2641 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
2701 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
2761 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
2821 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
2881 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
2941 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
3001 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt
3061 tgagtgagct gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga
3121 ggaagcggaa gagcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca
3181 ccgcaatggt gcactctcag tacaatctgc tctgatgccg catagttaag ccagtataca
3241 ctccgctatc gctacgtgac tgggtcatgg ctgcgccccg acacccgcca acacccgctg
3301 acgcgccctg acgggcttgt ctgctcccgg catccgctta cagacaagct gtgaccgtct
3361 ccgggagctg catgtgtcag aggttttcac cgtcatcacc gaaacgcgcg aggcagctgc
3421 ggtaaagctc atcagcgtgg tcgtgaagcg attcacagat gtctgcctgt tcatccgcgt
3481 ccagctcgtt gagcttctcc agaagcgtta atgtctggct tctgataaag cgggccatgt
3541 taagggcggt tttttcctgt ttggtcactg atgcctccgt gtaaggggga tttctgttca
3601 tgggggtaat gataccgatg aaacgagaga ggatgctcac gatacgggtt actgatgatg
3661 aacatgcccg gttactggaa cgttgtgagg gtaaacaact ggcggtatgg atgcggcggg
3721 accagagaaa aatcactcag ggtcaatgcc agcgcttcgt taatacagat gtaggtgttc
3781 cacagggtag ccagcagcat cctgcgatgc agatccggaa cataatggtg cagggcgctg
3841 acttccgcgt ttccagactt tacgaaacac ggaaaccgaa gaccattcat gttgttgctc
3901 aggtcgcaga cgttttgcag cagcagtcgc ttcacgttcg ctcgcgtatc ggtgattcat
3961 tctgctaacc agtaaggcaa ccccgccagc ctagccgggt cctcaacgac aggagcacga
4021 tcatgcgcac ccgtggccag gacccaacgc tgcccgagat ctcgatcccg cgaaat
//
