SpyTag003-mKate2

LOCUS       SpyTag003-mKate2                6024 bp ds-DNA     

circular SYN 12-MAY-2021
DEFINITION  Expresses SpyTag003-mKate2 (a far-red fluorescent 

protein) in 
            bacterial cytoplasm.
ACCESSION   .
VERSION     .
KEYWORDS    SpyTag003-mKate2
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6024)
  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 6024)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.biofeng.com
FEATURES             Location/Qualifiers
     source          1..6024
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     RBS             15..37
                     /note="efficient ribosome binding site from 

bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             117..809
                     /codon_start=1
                     /product="monomeric far-red fluorescent 

protein (Shcherbo 
                     et al., 2009)"
                     /label=mKate2
                     /note="mammalian codon-optimized"
                     

/translation="VSELIKENMHMKLYMEGTVNNHHFKCTSEGEGKPYEGTQTMRIKA
                     

VEGGPLPFAFDILATSFMYGSKTFINHTQGIPDFFKQSFPEGFTWERVTTYEDGGVLTA
                     

TQDTSLQDGCLIYNVKIRGVNFPSNGPVMQKKTLGWEASTETLYPADGGLEGRADMALK
                     

LVGGGHLICNLKTTYRSKKPAKNLKMPGVYYVDRRLERIKEADKETYVEQHEVAVARYC
                     DLPSKLGHR"
     CDS             828..845
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(915..933)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      929..976
                     /label=T7 terminator
                     /note="transcription terminator for 

bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1013..1468
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of 

replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1100..1119)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1310..1331
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1560..2375)
                     /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     2283..2302
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse 

primer"
     primer_bind     2367..2386
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2497..3085
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC 

origin of 
                     replication"
     primer_bind     2986..3005
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3239..3256
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3271..3410
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3336..3355)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast 

selectable 
                     marker"
     primer_bind     3496..3518
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3512..3703)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains 

plasmids at low copy
                     number"
                     /label=rop
                     

/translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4512..5594)
                     /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     5556..5575
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5595..5672)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5878..5897
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream 

of BamHI, 
                     reverse primer"
     primer_bind     5981..6000
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5981..5999
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA 

polymerase"
     protein_bind    6000..6024
                     /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)."
ORIGIN
        1 cctctagaaa taattttgtt taactttaag aaggagatat acatatggga 

cgtggcgttc
       61 ctcatattgt tatggtggac gcctacaaac gctataaatc gggaggtggt 

tcaggcgtga
      121 gcgagctgat taaggagaac atgcacatga agctgtacat ggagggcacc 

gtgaacaacc
      181 accacttcaa gtgcacatcc gagggcgaag gcaagcccta cgagggcacc 

cagaccatga
      241 gaatcaaggc cgtcgagggc ggccctctcc ccttcgcctt cgacatcctg 

gctaccagct
      301 tcatgtacgg cagcaaaacc ttcatcaacc acacccaggg catccccgac 

ttctttaagc
      361 agtccttccc tgagggcttc acatgggaga gagtcaccac atacgaagac 

gggggcgtgc
      421 tgaccgctac ccaggacacc agcctccagg acggctgcct catctacaac 

gtcaagatca
      481 gaggggtgaa cttcccatcc aacggccctg tgatgcagaa gaaaacactc 

ggctgggagg
      541 cctccaccga gaccctgtac cccgctgacg gcggcctgga aggcagagcc 

gacatggccc
      601 tgaagctcgt gggcgggggc cacctgatct gcaacttgaa gaccacatac 

agatccaaga
      661 aacccgctaa gaacctcaag atgcccggcg tctactatgt ggacagaaga 

ctggaaagaa
      721 tcaaggaggc cgacaaagag acctacgtcg agcagcacga ggtggctgtg 

gccagatact
      781 gcgacctccc tagcaaactg gggcacagag gctccggctc cggctcccac 

catcaccatc
      841 accattgata agctagcacc actgagatcc ggctgctaac aaagcccgaa 

aggaagctga
      901 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct 

ctaaacgggt
      961 cttgaggggt tttttgctga aaggaggaac tatatccgga ttggcgaatg 

ggacgcgccc
     1021 tgtagcggcg cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac 

cgctacactt
     1081 gccagcgccc tagcgcccgc tcctttcgct ttcttccctt cctttctcgc 

cacgttcgcc
     1141 ggctttcccc gtcaagctct aaatcggggg ctccctttag ggttccgatt 

tagtgcttta
     1201 cggcacctcg accccaaaaa acttgattag ggtgatggtt cacgtagtgg 

gccatcgccc
     1261 tgatagacgg tttttcgccc tttgacgttg gagtccacgt tctttaatag 

tggactcttg
     1321 ttccaaactg gaacaacact caaccctatc tcggtctatt cttttgattt 

ataagggatt
     1381 ttgccgattt cggcctattg gttaaaaaat gagctgattt aacaaaaatt 

taacgcgaat
     1441 tttaacaaaa tattaacgct tacaatttag gtggcacttt tcggggaaat 

gtgcgcggaa
     1501 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg 

aattaattct
     1561 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg 

attatcaata
     1621 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag 

gcagttccat
     1681 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc 

aatacaacct
     1741 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg 

agtgacgact
     1801 gaatccggtg agaatggcaa aagtttatgc atttctttcc agacttgttc 

aacaggccag
     1861 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat 

tcgtgattgc
     1921 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac 

aggaatcgaa
     1981 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga 

atcaggatat
     2041 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa 

ccatgcatca
     2101 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt 

cagccagttt
     2161 agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg 

tttcagaaac
     2221 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga 

ttgcccgaca
     2281 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt 

taatcgcggc
     2341 ctagagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt 

actgtttatg
     2401 taagcagaca gttttattgt tcatgaccaa aatcccttaa cgtgagtttt 

cgttccactg
     2461 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt 

ttctgcgcgt
     2521 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt 

tgccggatca
     2581 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga 

taccaaatac
     2641 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag 

caccgcctac
     2701 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata 

agtcgtgtct
     2761 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg 

gctgaacggg
     2821 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga 

gatacctaca
     2881 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca 

ggtatccggt
     2941 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa 

acgcctggta
     3001 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt 

tgtgatgctc
     3061 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac 

ggttcctggc
     3121 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt 

ctgtggataa
     3181 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga 

ccgagcgcag
     3241 cgagtcagtg agcgaggaag cggaagagcg cctgatgcgg tattttctcc 

ttacgcatct
     3301 gtgcggtatt tcacaccgca atggtgcact ctcagtacaa tctgctctga 

tgccgcatag
     3361 ttaagccagt atacactccg ctatcgctac gtgactgggt catggctgcg 

ccccgacacc
     3421 cgccaacacc cgctgacgcg ccctgacggg cttgtctgct cccggcatcc 

gcttacagac
     3481 aagctgtgac cgtctccggg agctgcatgt gtcagaggtt ttcaccgtca 

tcaccgaaac
     3541 gcgcgaggca gctgcggtaa agctcatcag cgtggtcgtg aagcgattca 

cagatgtctg
     3601 cctgttcatc cgcgtccagc tcgttgagtt tctccagaag cgttaatgtc 

tggcttctga
     3661 taaagcgggc catgttaagg gcggtttttt cctgtttggt cactgatgcc 

tccgtgtaag
     3721 ggggatttct gttcatgggg gtaatgatac cgatgaaacg agagaggatg 

ctcacgatac
     3781 gggttactga tgatgaacat gcccggttac tggaacgttg tgagggtaaa 

caactggcgg
     3841 tatggatgcg gcgggaccag agaaaaatca ctcagggtca atgccagcgc 

ttcgttaata
     3901 cagatgtagg tgttccacag ggtagccagc agcatcctgc gatgcagatc 

cggaacataa
     3961 tggtgcaggg cgctgacttc cgcgtttcca gactttacga aacacggaaa 

ccgaagacca
     4021 ttcatgttgt tgctcaggtc gcagacgttt tgcagcagca gtcgcttcac 

gttcgctcgc
     4081 gtatcggtga ttcattctgc taaccagtaa ggcaaccccg ccagcctagc 

cgggtcctca
     4141 acgacaggag cacgatcatg cgcacccgtg gggccgccat gccggcgata 

atggcctgct
     4201 tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg 

gcgtgcaaga
     4261 ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag 

cggtcctcgc
     4321 cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata 

aagaagacag
     4381 tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg 

actgggttga
     4441 aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa 

cttacattaa
     4501 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag 

ctgcattaat
     4561 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt 

ggtttttctt
     4621 ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg 

agagagttgc
     4681 agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat 

ggtggttaac
     4741 ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga 

gatatccgca
     4801 ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat 

ctgatcgttg
     4861 gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt 

ttgttgaaaa
     4921 ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg 

attgcgagtg
     4981 agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa 

tgggcccgct
     5041 aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag 

tcgcgtaccg
     5101 tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc 

aagaaataac
     5161 gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc 

cagcggatag
     5221 ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc 

tttacaggct
     5281 tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg 

atcggcgcga
     5341 gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga 

ggtggcaacg
     5401 ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg 

aatgtaattc
     5461 agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg 

gctggcctgg
     5521 ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac 

atcgtataac
     5581 gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta 

tcatgccata
     5641 ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc 

ccttatgcga
     5701 ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg 

ccgccgcaag
     5761 gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc 

ctgccaccat
     5821 acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt 

ccccatcggt
     5881 gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc 

cggccacgat
     5941 gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact 

cactataggg
     6001 gaattgtgag cggataacaa ttcc
//