LOCUS       sequence-183106-        6791 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression plasmid containing SpyCatcher-ELP-GFP fusion. 
            SpyCatcher forms a covalent bond with SpyTag and can be used to 
            label plasma membrane localized SpyTag-C1C2 in live cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6791)
  AUTHORS   Bedbrook CN, Kato M, Ravindra Kumar S, Lakshmanan A, Nath RD, Sun F,
            Sternberg PW, Arnold FH, Gradinaru V
  TITLE     Genetically Encoded Spy Peptide Fusion System to Detect Plasma 
            Membrane-Localized Proteins In Vivo.
  JOURNAL   Chem Biol. 2015 Aug 20;22(8):1108-21. doi: 
            10.1016/j.chembiol.2015.06.020. Epub 2015 Jul 23.
  PUBMED    26211362
REFERENCE   2  (bases 1 to 6791)
  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"; doi: 
            "10.1016/j.chembiol.2015.06.020"; journalName: "Chem Biol"; date: 
            "2015-08-20- 20"; volume: "22"; issue: "8"; pages: "1108-21"
COMMENT     Sequence Label: pQE80L-SpyCatcher-ELP-GFP
FEATURES             Location/Qualifiers
     source          1..6791
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(10..24,25..30,31..47,48..54)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    30..46
                     /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)."
     protein_bind    62..78
                     /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             97..108
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             130..147
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             166..186
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             193..531
                     /codon_start=1
                     /product="truncated version of the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyCatcher
                     /note="forms a covalent isopeptide bond with the SpyTag 
                     peptide (Zakeri et al., 2012)"
                     /translation="VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATME
                     LRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     KATKGDAHI"
     CDS             1792..1812
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1819..2157
                     /codon_start=1
                     /product="truncated version of the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyCatcher
                     /note="forms a covalent isopeptide bond with the SpyTag 
                     peptide (Zakeri et al., 2012)"
                     /translation="VDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATME
                     LRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNG
                     KATKGDAHI"
     terminator      2248..2342
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2386..3045
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2452..2471)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      3110..3196
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3289..4371)
                     /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     4333..4352
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4372..4449)
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     primer_bind     complement(4533..4555)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4641..4781
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4696..4715
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4796..4813)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4967..5555)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5047..5066)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5726..6517,6518..6586))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6349..6368
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6587..6691)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6759..6777
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 cttgagaaat cataaaaaat ttatttgctt tgtgagcgga taacaattat aatagattca
       61 attgtgagcg gataacaatt tcacacagaa ttcattaaag aggagaaatt aactatgaaa
      121 ggcagcagcc atcatcatca tcatcacgtc gacatcccaa cgaccgaaaa cctgtatttt
      181 cagggcgcca tggttgatac cttatcaggt ttatcaagtg agcaaggtca gtccggtgat
      241 atgacaattg aagaagatag tgctacccat attaaattct caaaacgtga tgaggacggc
      301 aaagagttag ctggtgcaac tatggagttg cgtgattcat ctggtaaaac tattagtaca
      361 tggatttcag atggacaagt gaaagatttc tacctgtatc caggaaaata tacatttgtc
      421 gaaaccgcag caccagacgg ttatgaggta gcaactgcta ttacctttac agttaatgag
      481 caaggtcagg ttactgtaaa tggcaaagca actaaaggtg acgctcatat tgacggtccg
      541 caaggtattt ggggtcagct cgacggccac ggcgtgggtg ttccgggcgt cggtgtgccg
      601 ggtgtgggtg tgccgggcga aggtgtgccg ggcgtcggtg tgccgggtgt tggtgttccg
      661 ggcgttggtg tgccgggcgt tggcgtgccg ggcgagggtg tgccgggcgt tggtgttccg
      721 ggcgtgggtg tgccgggcgt gggcgtgccg ggcgtcggtg ttccgggcga gggtgtgccg
      781 ggcgtaggtg tgccgggcgt tggtgagctc atgagtaaag gagaagaact tttcactgga
      841 gtcgtcccaa ttcttgttga attagatggt gatgttaatg ggcacaaatt ttctgtcaga
      901 ggagagggtg aaggtgatgc cacatacgga aaaattaccc ttaaattgat ttgcactact
      961 ggaaaactac ctgttccatg gccaacactt gtcactactt gcggttatgg tgttcaatgc
     1021 tttgcgcgtt atccggatca tctgaaacgg catgactttt tcaagagtgc ctttcccgaa
     1081 ggttatgtac aggaacgcac tatatctttc aaagatgacg ggaagttcaa gacgcgtgct
     1141 gaagtcaagt ttgaaggtga taccattgtt aatcgtatca agttaaaagg cattgatttt
     1201 aaagaagatg gaaacattct cggacacaaa ctcgagtaca actataactc acacgatgta
     1261 tacatcacgg cagacaaaca aaagactgga atcaaagcta acttcaaaat tcgccacaac
     1321 gttgaagatg gttccgttca actggcagac cattatcaac aaaatactcc aattggcgat
     1381 ggccctgtcc gtttaccaga caaccattac ctgttgacac aatctgtcat ttcgaaagat
     1441 cccaacgaaa agcgtgacca cgcggtcctt catgagtttg taactgctgc tgggattaca
     1501 catggcatcg atgaactcta caaataaact agtgtgccgg gcgtcggcgt gccgggcgta
     1561 ggtgttccgg gcgagggtgt tccgggcgtt ggtgtgccgg gcgtcggcgt gccgggcgtg
     1621 ggtgttccgg gcgtaggtgt gccgggcgag ggtgtgccgg gcgtgggcgt gccgggcgta
     1681 ggtgttccgg gcgtaggtgt tccgggcgta ggtgttccgg gtgaaggcgt gccgggcgtt
     1741 ggtgtgccgg gtgtgggcgt gccgggcggg ctgctcgaca tcccaacgac cgaaaacctg
     1801 tattttcagg gcgccatggt tgatacctta tcaggtttat caagtgagca aggtcagtcc
     1861 ggtgatatga caattgaaga agatagtgct acccatatta aattctcaaa acgtgatgag
     1921 gacggcaaag agttagctgg tgcaactatg gagttgcgtg attcatctgg taaaactatt
     1981 agtacatgga tttcagatgg acaagtgaaa gatttctacc tgtatccagg aaaatataca
     2041 tttgtcgaaa ccgcagcacc agacggttat gaggtagcaa ctgctattac ctttacagtt
     2101 aatgagcaag gtcaggttac tgtaaatggc aaagcaacta aaggtgacgc tcatattgac
     2161 ggtccgcaag gtatttgggg tcagctcgag tggaagaaat aaggatccgg ctgctaacaa
     2221 agcccgaagc ttaattagct gagcttggac tcctgttgat agatccagta atgacctcag
     2281 aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt attggtgaga
     2341 atccaagcca gcttggcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc
     2401 actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt
     2461 cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta
     2521 aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc
     2581 ctgatgaatg ctcatccgga atttcgtatg gcaatgaaag acggtgagct ggtgatatgg
     2641 gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc
     2701 tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg
     2761 tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc
     2821 tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac
     2881 ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg
     2941 ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt
     3001 aatgaattac aacagtactg cgatgagtgg cagggcgggg cgtaattttt ttaaggcagt
     3061 tattggtgcc cttaaacgcc tggggtaatg actctctagc ttgaggcatc aaataaaacg
     3121 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct
     3181 cctgagtagg acaaatccgc cctctagatt acgtgcagtc gatgataagc tgtcaaacat
     3241 gagaattgtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     3301 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     3361 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     3421 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     3481 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     3541 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     3601 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     3661 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     3721 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     3781 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     3841 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     3901 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     3961 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     4021 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     4081 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     4141 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     4201 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     4261 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     4321 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     4381 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcaccattc
     4441 gatggtgtcg gaatttcggg cagcgttggg tcctggccac gggtgcgcat gatctagagc
     4501 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     4561 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     4621 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat
     4681 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
     4741 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
     4801 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4861 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4921 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4981 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5041 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5101 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5161 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5221 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5281 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5341 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5401 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5461 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5521 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5581 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5641 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5701 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5761 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5821 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5881 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5941 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6001 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6061 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6121 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6181 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6241 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6301 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6361 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6421 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6481 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6541 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6601 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6661 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     6721 tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc acgaggccct
     6781 ttcgtcttca c
//
