LOCUS       sequence-221422-        6539 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Two SpyCatchers linked by an elastin-like polypeptide with a 
            C-terminal SnoopCatcher.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6539)
  AUTHORS   Wieduwild R, Howarth M
  TITLE     Assembling and decorating hyaluronan hydrogels with twin protein 
            superglues to mimic cell-cell interactions.
  JOURNAL   Biomaterials. 2018 Oct;180:253-264. doi: 
            10.1016/j.biomaterials.2018.07.020. Epub 2018 Jul 17.
  PUBMED    30053659
REFERENCE   2  (bases 1 to 6539)
  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: 
            "Biomaterials."; date: "2018-10"; volume: "180:253-264. doi"; pages:
            " 10.1016/j.biomaterials.2018.07.020. Epub 2018 Jul 17"
COMMENT     Sequence Label: pQE80L TriCatcher-ELP
FEATURES             Location/Qualifiers
     source          1..6539
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     40..58
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(82..96,97..102,103..119,120..126)
                     /label=T5 promoter
                     /note="bacteriophage T5 promoter for E. coli RNA 
                     polymerase, with embedded lac operator"
     protein_bind    102..118
                     /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    134..150
                     /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             169..180
                     /note="strong bacterial ribosome binding site (Elowitz and 
                     Leibler, 2000)"
     CDS             202..219
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             238..258
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             265..603
                     /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             1198..1218
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1225..1563
                     /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             1684..2019
                     /label=SnoopCatcher
     terminator      2068..2162
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2206..2865
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2272..2291)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2930..3016
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3109..4191)
                     /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     4153..4172
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4192..4269)
                     /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(4353..4375)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4461..4601
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4516..4535
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4616..4633)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4787..5375)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4867..4886)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5546..6337,6338..6406))
                     /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     6169..6188
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6407..6511)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtctaagaaa ccattattat catgacatta acctataaaa ataggcgtat cacgaggccc
       61 tttcgtcttc accttgagaa atcataaaaa atttatttgc tttgtgagcg gataacaatt
      121 ataatagatt caattgtgag cggataacaa tttcacacag aattcattaa agaggagaaa
      181 ttaactatga aaggcagcag ccatcatcat catcatcacg tcgacatccc aacgaccgaa
      241 aacctgtatt ttcagggcgc catggttgat accttatcag gtttatcaag tgagcaaggt
      301 cagtccggtg atatgacaat tgaagaagat agtgctaccc atattaaatt ctcaaaacgt
      361 gatgaggacg gcaaagagtt agctggtgca actatggagt tgcgtgattc atctggtaaa
      421 actattagta catggatttc agatggacaa gtgaaagatt tctacctgta tccaggaaaa
      481 tatacatttg tcgaaaccgc agcaccagac ggttatgagg tagcaactgc tattaccttt
      541 acagttaatg agcaaggtca ggttactgta aatggcaaag caactaaagg tgacgctcat
      601 attgacggta gcggtggctc tggcggtcag ctcgacggcc acggcgtggg tgttccgggc
      661 gtcggtgtgc cgggtgtggg tgtgccgggc gaaggtgtgc cgggcgtcgg tgtgccgggt
      721 gttggtgttc cgggcgttgg tgtgccgggc gttggcgtgc cgggcgaggg tgtgccgggc
      781 gttggtgttc cgggcgtggg tgtgccgggc gtgggcgtgc cgggcgtcgg tgttccgggc
      841 gagggtgtgc cgggcgtagg tgtgccgggc gttggtgagc tctatgcggt taccggtcgc
      901 ggtcgcgccc cggccagctc tgccccgatc gccactagtg tgccgggcgt cggcgtgccg
      961 ggcgtaggtg ttccgggcga gggtgttccg ggcgttggtg tgccgggcgt cggcgtgccg
     1021 ggcgtgggtg ttccgggcgt aggtgtgccg ggcgagggtg tgccgggcgt gggcgtgccg
     1081 ggcgtaggtg ttccgggcgt aggtgttccg ggcgtaggtg ttccgggtga aggcgtgccg
     1141 ggcgttggtg tgccgggtgt gggcgtgccg ggcgggctgc tcgacatccc aacgaccgaa
     1201 aacctgtatt ttcagggcgc catggttgat accttatcag gtttatcaag tgagcaaggt
     1261 cagtccggtg atatgacaat tgaagaagat agtgctaccc atattaaatt ctcaaaacgt
     1321 gatgaggacg gcaaagagtt agctggtgca actatggagt tgcgtgattc atctggtaaa
     1381 actattagta catggatttc agatggacaa gtgaaagatt tctacctgta tccaggaaaa
     1441 tatacatttg tcgaaaccgc agcaccagac ggttatgagg tagcaactgc tattaccttt
     1501 acagttaatg agcaaggtca ggttactgta aatggcaaag caactaaagg tgacgctcat
     1561 attggatccc ccgccaacct gaaggccctg gaggcccaga agcagaagga gcagagacag
     1621 gccgccgagg agctggccaa cgccaagaag ctgaaggagc agctggagaa gggatcccat
     1681 atgaagccgc tgcgtggtgc cgtgtttagc ctgcagaaac agcatcccga ctatcccgat
     1741 atctatggcg cgattgatca gaatgggacc tatcaaaatg tgcgtaccgg cgaagatggt
     1801 aaactgacct ttaagaatct gagcgatggc aaatatcgcc tgtttgaaaa tagcgaaccc
     1861 gctggctata aaccggtgca gaataagccg attgtggcgt ttcagattgt gaatggcgaa
     1921 gtgcgtgatg tgaccagcat tgtgccgcag gatattccgg ctacatatga atttaccaac
     1981 ggtaaacatt atatcaccaa tgaaccgata ccgccgaaat aaggatccgg ctgctaacaa
     2041 agcccgaagc ttaattagct gagcttggac tcctgttgat agatccagta atgacctcag
     2101 aactccatct ggatttgttc agaacgctcg gttgccgccg ggcgtttttt attggtgaga
     2161 atccaagcca gcttggcgag attttcagga gctaaggaag ctaaaatgga gaaaaaaatc
     2221 actggatata ccaccgttga tatatcccaa tggcatcgta aagaacattt tgaggcattt
     2281 cagtcagttg ctcaatgtac ctataaccag accgttcagc tggatattac ggccttttta
     2341 aagaccgtaa agaaaaataa gcacaagttt tatccggcct ttattcacat tcttgcccgc
     2401 ctgatgaatg ctcatccgga atttcgtatg gcaatgaaag acggtgagct ggtgatatgg
     2461 gatagtgttc acccttgtta caccgttttc catgagcaaa ctgaaacgtt ttcatcgctc
     2521 tggagtgaat accacgacga tttccggcag tttctacaca tatattcgca agatgtggcg
     2581 tgttacggtg aaaacctggc ctatttccct aaagggttta ttgagaatat gtttttcgtc
     2641 tcagccaatc cctgggtgag tttcaccagt tttgatttaa acgtggccaa tatggacaac
     2701 ttcttcgccc ccgttttcac catgggcaaa tattatacgc aaggcgacaa ggtgctgatg
     2761 ccgctggcga ttcaggttca tcatgccgtt tgtgatggct tccatgtcgg cagaatgctt
     2821 aatgaattac aacagtactg cgatgagtgg cagggcgggg cgtaattttt ttaaggcagt
     2881 tattggtgcc cttaaacgcc tggggtaatg actctctagc ttgaggcatc aaataaaacg
     2941 aaaggctcag tcgaaagact gggcctttcg ttttatctgt tgtttgtcgg tgaacgctct
     3001 cctgagtagg acaaatccgc cctctagatt acgtgcagtc gatgataagc tgtcaaacat
     3061 gagaattgtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     3121 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     3181 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     3241 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     3301 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     3361 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     3421 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     3481 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     3541 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     3601 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     3661 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     3721 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     3781 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     3841 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     3901 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     3961 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     4021 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     4081 tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg aaacggtctg
     4141 ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca cattcaccac
     4201 cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt tgcaccattc
     4261 gatggtgtcg gaatttcggg cagcgttggg tcctggccac gggtgcgcat gatctagagc
     4321 tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct cccggagacg
     4381 gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg cgcgtcagcg
     4441 ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag cggagtgtat
     4501 actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat atgcggtgtg
     4561 aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc gcttcctcgc
     4621 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     4681 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     4741 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     4801 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     4861 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     4921 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     4981 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5041 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5101 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5161 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5221 ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5281 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5341 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5401 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5461 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5521 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     5581 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     5641 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     5701 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     5761 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     5821 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     5881 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     5941 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6001 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6061 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6121 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6181 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6241 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6301 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6361 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     6421 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     6481 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgac
//
