LOCUS       sequence-387266-        6692 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression of SpyCatcher-ELP-CarHC-ELP-SpyCatcher, with TEV 
            cleavage site and MMP cleavage site respectively before and after 
            each SpyCatcher.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6692)
  AUTHORS   Wang R, Yang Z, Luo J, Hsing IM, Sun F
  TITLE     B(12)-dependent photoresponsive protein hydrogels for controlled 
            stem cell/protein release.
  JOURNAL   Proc Natl Acad Sci U S A. 2017 Jun 6;114(23):5912-5917. doi: 
            10.1073/pnas.1621350114. Epub 2017 May 22.
  PUBMED    28533376
REFERENCE   2  (bases 1 to 6692)
  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: 
            "10.1073/pnas.1621350114"; journalName: "Proc Natl Acad Sci U S A"; 
            date: "2017-06-6- 6"; volume: "114"; issue: "23"; pages: "5912-5917"
COMMENT     Sequence Label: pQE80l BCB
FEATURES             Location/Qualifiers
     source          1..6692
                     /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             1765..1785
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1792..2130
                     /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      2221..2315
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2359..3018
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2425..2444)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      3083..3169
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3262..4344)
                     /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     4306..4325
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4345..4422)
                     /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(4506..4528)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4614..4754
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4669..4688
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4769..4786)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4940..5528)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5020..5039)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5699..6490,6491..6559))
                     /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     6322..6341
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6560..6664)
                     /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 attgacggtc cgcaaggtat ttggggtcag 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 tcccagaaga tctgggcacc
      901 ggcctgctgg aagcactgct gcgcggtgat ctggcgggcg ccgaagctct gtttcgtcgt
      961 ggcctgcgtt tctggggccc ggaaggcgtt ctggagcacc tgctgctgcc ggtgctgcgt
     1021 gaagtgggcg aagcttggca ccgtggtgaa atcggcgttg cagaagaaca cctggcgagc
     1081 accttcctgc gcgcgcgtct gcaggagcta ctggacctgg caggtttccc gccgggtccg
     1141 ccggtcctgg tgactacgcc gccgggcgaa cgccacgaaa tcggtgcgat gctggcggcg
     1201 taccatctgc gtcgtaaggg cgtcccggcg ctgtatctgg gcccggatac tccgctgccg
     1261 gacctgcgtg cactggcgcg ccgcctgggt gcaggcgcgg tcgtgctgtc tgctgttctg
     1321 agcgaaccgc tgcgtgctct gcctgacggt gccctgaaag atctggcacc gcgtgttttc
     1381 ctgggcggcc agggcgcagg cccggaagag gcacgccgtc tgggtgccga atacatggaa
     1441 gacctgaaag gcctggctga agcgctgtgg ctgccgcgcg gtccggaaaa agaagcaatc
     1501 actagtgtgc cgggcgtcgg cgtgccgggc gtaggtgttc cgggcgaggg tgttccgggc
     1561 gttggtgtgc cgggcgtcgg cgtgccgggc gtgggtgttc cgggcgtagg tgtgccgggc
     1621 gagggtgtgc cgggcgtggg cgtgccgggc gtaggtgttc cgggcgtagg tgttccgggc
     1681 gtaggtgttc cgggtgaagg cgtgccgggc gttggtgtgc cgggtgtggg cgtgccgggc
     1741 gggctgctcg acatcccaac gaccgaaaac ctgtattttc agggcgccat ggttgatacc
     1801 ttatcaggtt tatcaagtga gcaaggtcag tccggtgata tgacaattga agaagatagt
     1861 gctacccata ttaaattctc aaaacgtgat gaggacggca aagagttagc tggtgcaact
     1921 atggagttgc gtgattcatc tggtaaaact attagtacat ggatttcaga tggacaagtg
     1981 aaagatttct acctgtatcc aggaaaatat acatttgtcg aaaccgcagc accagacggt
     2041 tatgaggtag caactgctat tacctttaca gttaatgagc aaggtcaggt tactgtaaat
     2101 ggcaaagcaa ctaaaggtga cgctcatatt gacggtccgc aaggtatttg gggtcagctc
     2161 gagtggaaga aataaggatc cggctgctaa caaagcccga agcttaatta gctgagcttg
     2221 gactcctgtt gatagatcca gtaatgacct cagaactcca tctggatttg ttcagaacgc
     2281 tcggttgccg ccgggcgttt tttattggtg agaatccaag ccagcttggc gagattttca
     2341 ggagctaagg aagctaaaat ggagaaaaaa atcactggat ataccaccgt tgatatatcc
     2401 caatggcatc gtaaagaaca ttttgaggca tttcagtcag ttgctcaatg tacctataac
     2461 cagaccgttc agctggatat tacggccttt ttaaagaccg taaagaaaaa taagcacaag
     2521 ttttatccgg cctttattca cattcttgcc cgcctgatga atgctcatcc ggaatttcgt
     2581 atggcaatga aagacggtga gctggtgata tgggatagtg ttcacccttg ttacaccgtt
     2641 ttccatgagc aaactgaaac gttttcatcg ctctggagtg aataccacga cgatttccgg
     2701 cagtttctac acatatattc gcaagatgtg gcgtgttacg gtgaaaacct ggcctatttc
     2761 cctaaagggt ttattgagaa tatgtttttc gtctcagcca atccctgggt gagtttcacc
     2821 agttttgatt taaacgtggc caatatggac aacttcttcg cccccgtttt caccatgggc
     2881 aaatattata cgcaaggcga caaggtgctg atgccgctgg cgattcaggt tcatcatgcc
     2941 gtttgtgatg gcttccatgt cggcagaatg cttaatgaat tacaacagta ctgcgatgag
     3001 tggcagggcg gggcgtaatt tttttaaggc agttattggt gcccttaaac gcctggggta
     3061 atgactctct agcttgaggc atcaaataaa acgaaaggct cagtcgaaag actgggcctt
     3121 tcgttttatc tgttgtttgt cggtgaacgc tctcctgagt aggacaaatc cgccctctag
     3181 attacgtgca gtcgatgata agctgtcaaa catgagaatt gtgcctaatg agtgagctaa
     3241 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     3301 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     3361 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
     3421 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
     3481 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
     3541 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
     3601 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
     3661 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
     3721 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
     3781 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
     3841 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
     3901 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
     3961 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
     4021 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
     4081 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
     4141 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
     4201 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
     4261 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
     4321 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     4381 tcatgccata ccgcgaaagg ttttgcacca ttcgatggtg tcggaatttc gggcagcgtt
     4441 gggtcctggc cacgggtgcg catgatctag agctgcctcg cgcgtttcgg tgatgacggt
     4501 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     4561 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     4621 atgacccagt cacgtagcga tagcggagtg tatactggct taactatgcg gcatcagagc
     4681 agattgtact gagagtgcac catatgcggt gtgaaatacc gcacagatgc gtaaggagaa
     4741 aataccgcat caggcgctct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
     4801 ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
     4861 gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa
     4921 aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
     4981 gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
     5041 ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg
     5101 cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
     5161 cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
     5221 gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc
     5281 cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
     5341 agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt ggtatctgcg
     5401 ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa
     5461 ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
     5521 gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
     5581 cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa
     5641 attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
     5701 accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
     5761 ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca tctggcccca
     5821 gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca gcaataaacc
     5881 agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc tccatccagt
     5941 ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg
     6001 ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg gcttcattca
     6061 gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc aaaaaagcgg
     6121 ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg ttatcactca
     6181 tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg
     6241 tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct
     6301 cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca
     6361 tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca
     6421 gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg
     6481 tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac
     6541 ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt
     6601 attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc
     6661 cgcgcacatt tccccgaaaa gtgccacctg ac
//
