LOCUS       sequence-222151-        6545 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Two SpyCatchers linked by an elastin-like polypeptide with an RGDSP 
            integrin-binding site, an MMP-cleavable sequence and a C-terminal 
            SnoopCatcher.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6545)
  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 6545)
  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-RGDSP-MMP
FEATURES             Location/Qualifiers
     source          1..6545
                     /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             1204..1224
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1231..1569
                     /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             1690..2025
                     /label=SnoopCatcher
     terminator      2074..2168
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2212..2871
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2278..2297)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2936..3022
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3115..4197)
                     /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     4159..4178
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4198..4275)
                     /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(4359..4381)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4467..4607
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4522..4541
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4622..4639)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4793..5381)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4873..4892)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5552..6343,6344..6412))
                     /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     6175..6194
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6413..6517)
                     /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 tctatgcgcg tggtgatagt
      901 ccggccgttc cgctcagtct ctatagtggt atccgtgcca ctagtgtgcc gggcgtcggc
      961 gtgccgggcg taggtgttcc gggcgagggt gttccgggcg ttggtgtgcc gggcgtcggc
     1021 gtgccgggcg tgggtgttcc gggcgtaggt gtgccgggcg agggtgtgcc gggcgtgggc
     1081 gtgccgggcg taggtgttcc gggcgtaggt gttccgggcg taggtgttcc gggtgaaggc
     1141 gtgccgggcg ttggtgtgcc gggtgtgggc gtgccgggcg ggctgctcga catcccaacg
     1201 accgaaaacc tgtattttca gggcgccatg gttgatacct tatcaggttt atcaagtgag
     1261 caaggtcagt ccggtgatat gacaattgaa gaagatagtg ctacccatat taaattctca
     1321 aaacgtgatg aggacggcaa agagttagct ggtgcaacta tggagttgcg tgattcatct
     1381 ggtaaaacta ttagtacatg gatttcagat ggacaagtga aagatttcta cctgtatcca
     1441 ggaaaatata catttgtcga aaccgcagca ccagacggtt atgaggtagc aactgctatt
     1501 acctttacag ttaatgagca aggtcaggtt actgtaaatg gcaaagcaac taaaggtgac
     1561 gctcatattg gatcccccgc caacctgaag gccctggagg cccagaagca gaaggagcag
     1621 agacaggccg ccgaggagct ggccaacgcc aagaagctga aggagcagct ggagaaggga
     1681 tcccatatga agccgctgcg tggtgccgtg tttagcctgc agaaacagca tcccgactat
     1741 cccgatatct atggcgcgat tgatcagaat gggacctatc aaaatgtgcg taccggcgaa
     1801 gatggtaaac tgacctttaa gaatctgagc gatggcaaat atcgcctgtt tgaaaatagc
     1861 gaacccgctg gctataaacc ggtgcagaat aagccgattg tggcgtttca gattgtgaat
     1921 ggcgaagtgc gtgatgtgac cagcattgtg ccgcaggata ttccggctac atatgaattt
     1981 accaacggta aacattatat caccaatgaa ccgataccgc cgaaataagg atccggctgc
     2041 taacaaagcc cgaagcttaa ttagctgagc ttggactcct gttgatagat ccagtaatga
     2101 cctcagaact ccatctggat ttgttcagaa cgctcggttg ccgccgggcg ttttttattg
     2161 gtgagaatcc aagccagctt ggcgagattt tcaggagcta aggaagctaa aatggagaaa
     2221 aaaatcactg gatataccac cgttgatata tcccaatggc atcgtaaaga acattttgag
     2281 gcatttcagt cagttgctca atgtacctat aaccagaccg ttcagctgga tattacggcc
     2341 tttttaaaga ccgtaaagaa aaataagcac aagttttatc cggcctttat tcacattctt
     2401 gcccgcctga tgaatgctca tccggaattt cgtatggcaa tgaaagacgg tgagctggtg
     2461 atatgggata gtgttcaccc ttgttacacc gttttccatg agcaaactga aacgttttca
     2521 tcgctctgga gtgaatacca cgacgatttc cggcagtttc tacacatata ttcgcaagat
     2581 gtggcgtgtt acggtgaaaa cctggcctat ttccctaaag ggtttattga gaatatgttt
     2641 ttcgtctcag ccaatccctg ggtgagtttc accagttttg atttaaacgt ggccaatatg
     2701 gacaacttct tcgcccccgt tttcaccatg ggcaaatatt atacgcaagg cgacaaggtg
     2761 ctgatgccgc tggcgattca ggttcatcat gccgtttgtg atggcttcca tgtcggcaga
     2821 atgcttaatg aattacaaca gtactgcgat gagtggcagg gcggggcgta atttttttaa
     2881 ggcagttatt ggtgccctta aacgcctggg gtaatgactc tctagcttga ggcatcaaat
     2941 aaaacgaaag gctcagtcga aagactgggc ctttcgtttt atctgttgtt tgtcggtgaa
     3001 cgctctcctg agtaggacaa atccgccctc tagattacgt gcagtcgatg ataagctgtc
     3061 aaacatgaga attgtgccta atgagtgagc taacttacat taattgcgtt gcgctcactg
     3121 cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg
     3181 gggagaggcg gtttgcgtat tgggcgccag ggtggttttt cttttcacca gtgagacggg
     3241 caacagctga ttgcccttca ccgcctggcc ctgagagagt tgcagcaagc ggtccacgct
     3301 ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt aacggcggga tataacatga
     3361 gctgtcttcg gtatcgtcgt atcccactac cgagatatcc gcaccaacgc gcagcccgga
     3421 ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg ttggcaacca gcatcgcagt
     3481 gggaacgatg ccctcattca gcatttgcat ggtttgttga aaaccggaca tggcactcca
     3541 gtcgccttcc cgttccgcta tcggctgaat ttgattgcga gtgagatatt tatgccagcc
     3601 agccagacgc agacgcgccg agacagaact taatgggccc gctaacagcg cgatttgctg
     3661 gtgacccaat gcgaccagat gctccacgcc cagtcgcgta ccgtcttcat gggagaaaat
     3721 aatactgttg atgggtgtct ggtcagagac atcaagaaat aacgccggaa cattagtgca
     3781 ggcagcttcc acagcaatgg catcctggtc atccagcgga tagttaatga tcagcccact
     3841 gacgcgttgc gcgagaagat tgtgcaccgc cgctttacag gcttcgacgc cgcttcgttc
     3901 taccatcgac accaccacgc tggcacccag ttgatcggcg cgagatttaa tcgccgcgac
     3961 aatttgcgac ggcgcgtgca gggccagact ggaggtggca acgccaatca gcaacgactg
     4021 tttgcccgcc agttgttgtg ccacgcggtt gggaatgtaa ttcagctccg ccatcgccgc
     4081 ttccactttt tcccgcgttt tcgcagaaac gtggctggcc tggttcacca cgcgggaaac
     4141 ggtctgataa gagacaccgg catactctgc gacatcgtat aacgttactg gtttcacatt
     4201 caccaccctg aattgactct cttccgggcg ctatcatgcc ataccgcgaa aggttttgca
     4261 ccattcgatg gtgtcggaat ttcgggcagc gttgggtcct ggccacgggt gcgcatgatc
     4321 tagagctgcc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg
     4381 gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg
     4441 tcagcgggtg ttggcgggtg tcggggcgca gccatgaccc agtcacgtag cgatagcgga
     4501 gtgtatactg gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
     4561 ggtgtgaaat accgcacaga tgcgtaagga gaaaataccg catcaggcgc tcttccgctt
     4621 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4681 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4741 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4801 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4861 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4921 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4981 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     5041 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     5101 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5161 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5221 gctacactag aaggacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5281 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5341 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5401 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5461 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5521 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5581 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5641 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5701 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5761 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5821 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5881 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5941 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     6001 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     6061 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6121 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6181 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6241 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6301 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6361 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6421 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6481 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6541 ctgac
//
