LOCUS       sequence-387241-        6377 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression of SpyCatcher-ELP-CarHCN-ELP-SpyCatcher, with TEV
            cleavage site and MMP cleavage site respectively before and after 
            each SpyCatcher, and GRGD site linked with (GGGGS)2 before CarHCN.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6377)
  AUTHORS   Yang Z, Fok HKF, Luo J, Yang Y, Wang R, Huang X, Sun F
  TITLE     B(12)-induced reassembly of split photoreceptor protein enables 
            photoresponsive hydrogels with tunable mechanics.
  JOURNAL   Sci Adv. 2022 Apr;8(13):eabm5482. doi: 10.1126/sciadv.abm5482. Epub 
            2022 Apr 1.
  PUBMED    35363531
REFERENCE   2  (bases 1 to 6377)
  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: "Sci Adv."; 
            date: "2022-04"; volume: "8(13):eabm5482. doi"; pages: " 
            10.1126/sciadv.abm5482. Epub 2022 Apr 1"
COMMENT     Sequence Label: pQE80l B-CN-B
FEATURES             Location/Qualifiers
     source          1..6377
                     /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             1450..1470
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1477..1815
                     /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      1906..2000
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2044..2703
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2110..2129)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2768..2854
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2947..4029)
                     /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     3991..4010
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4030..4107)
                     /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(4191..4213)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4299..4439
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4354..4373
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4454..4471)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4625..5213)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4705..4724)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5384..6175,6176..6244))
                     /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     6007..6026
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6245..6349)
                     /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 tcggccgtgg tgatggtggt
      901 gggggtagcg gtggtggggg tagtccagaa gatctgggca ccggcctgct ggaagcactg
      961 ctgcgcggtg atctggcggg cgccgaagct ctgtttcgtc gtggcctgcg tttctggggc
     1021 ccggaaggcg ttctggagca cctgctgctg ccggtgctgc gtgaagtggg cgaagcttgg
     1081 caccgtggtg aaatcggcgt tgcagaagaa cacctggcga gcaccttcct gcgcgcgcgt
     1141 ctgcaggagc tgctggacct ggcaggtttc ccgccgggtc cgccgactag tgtgccgggc
     1201 gtcggcgtgc cgggcgtagg tgttccgggc gagggtgttc cgggcgttgg tgtgccgggc
     1261 gtcggcgtgc cgggcgtggg tgttccgggc gtaggtgtgc cgggcgaggg tgtgccgggc
     1321 gtgggcgtgc cgggcgtagg tgttccgggc gtaggtgttc cgggcgtagg tgttccgggt
     1381 gaaggcgtgc cgggcgttgg tgtgccgggt gtgggcgtgc cgggcgggct gctcgacatc
     1441 ccaacgaccg aaaacctgta ttttcagggc gccatggttg ataccttatc aggtttatca
     1501 agtgagcaag gtcagtccgg tgatatgaca attgaagaag atagtgctac ccatattaaa
     1561 ttctcaaaac gtgatgagga cggcaaagag ttagctggtg caactatgga gttgcgtgat
     1621 tcatctggta aaactattag tacatggatt tcagatggac aagtgaaaga tttctacctg
     1681 tatccaggaa aatatacatt tgtcgaaacc gcagcaccag acggttatga ggtagcaact
     1741 gctattacct ttacagttaa tgagcaaggt caggttactg taaatggcaa agcaactaaa
     1801 ggtgacgctc atattgacgg tccgcaaggt atttggggtc agctcgagtg gaagaaataa
     1861 ggatccggct gctaacaaag cccgaagctt aattagctga gcttggactc ctgttgatag
     1921 atccagtaat gacctcagaa ctccatctgg atttgttcag aacgctcggt tgccgccggg
     1981 cgttttttat tggtgagaat ccaagccagc ttggcgagat tttcaggagc taaggaagct
     2041 aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa
     2101 gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg
     2161 gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt
     2221 attcacattc ttgcccgcct gatgaatgct catccggaat ttcgtatggc aatgaaagac
     2281 ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact
     2341 gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata
     2401 tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt
     2461 gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac
     2521 gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa
     2581 ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc
     2641 catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggcggggcg
     2701 taattttttt aaggcagtta ttggtgccct taaacgcctg gggtaatgac tctctagctt
     2761 gaggcatcaa ataaaacgaa aggctcagtc gaaagactgg gcctttcgtt ttatctgttg
     2821 tttgtcggtg aacgctctcc tgagtaggac aaatccgccc tctagattac gtgcagtcga
     2881 tgataagctg tcaaacatga gaattgtgcc taatgagtga gctaacttac attaattgcg
     2941 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     3001 ggccaacgcg cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac
     3061 cagtgagacg ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa
     3121 gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg
     3181 gatataacat gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac
     3241 gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac
     3301 cagcatcgca gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga
     3361 catggcactc cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata
     3421 tttatgccag ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag
     3481 cgcgatttgc tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc
     3541 atgggagaaa ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg
     3601 aacattagtg caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat
     3661 gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac
     3721 gccgcttcgt tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt
     3781 aatcgccgcg acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat
     3841 cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc
     3901 cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac
     3961 cacgcgggaa acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac
     4021 tggtttcaca ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg
     4081 aaaggttttg caccattcga tggtgtcgga atttcgggca gcgttgggtc ctggccacgg
     4141 gtgcgcatga tctagagctg cctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     4201 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     4261 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggcg cagccatgac ccagtcacgt
     4321 agcgatagcg gagtgtatac tggcttaact atgcggcatc agagcagatt gtactgagag
     4381 tgcaccatat gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     4441 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     4501 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     4561 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     4621 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     4681 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     4741 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     4801 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     4861 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     4921 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     4981 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     5041 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     5101 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     5161 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     5221 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     5281 tggtcatgag attatcaaaa aggatcttca cctagatcct tttaaattaa aaatgaagtt
     5341 ttaaatcaat ctaaagtata tatgagtaaa cttggtctga cagttaccaa tgcttaatca
     5401 gtgaggcacc tatctcagcg atctgtctat ttcgttcatc catagttgcc tgactccccg
     5461 tcgtgtagat aactacgata cgggagggct taccatctgg ccccagtgct gcaatgatac
     5521 cgcgagaccc acgctcaccg gctccagatt tatcagcaat aaaccagcca gccggaaggg
     5581 ccgagcgcag aagtggtcct gcaactttat ccgcctccat ccagtctatt aattgttgcc
     5641 gggaagctag agtaagtagt tcgccagtta atagtttgcg caacgttgtt gccattgcta
     5701 caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc attcagctcc ggttcccaac
     5761 gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc
     5821 ctccgatcgt tgtcagaagt aagttggccg cagtgttatc actcatggtt atggcagcac
     5881 tgcataattc tcttactgtc atgccatccg taagatgctt ttctgtgact ggtgagtact
     5941 caaccaagtc attctgagaa tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa
     6001 tacgggataa taccgcgcca catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt
     6061 cttcggggcg aaaactctca aggatcttac cgctgttgag atccagttcg atgtaaccca
     6121 ctcgtgcacc caactgatct tcagcatctt ttactttcac cagcgtttct gggtgagcaa
     6181 aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc gacacggaaa tgttgaatac
     6241 tcatactctt cctttttcaa tattattgaa gcatttatca gggttattgt ctcatgagcg
     6301 gatacatatt tgaatgtatt tagaaaaata aacaaatagg ggttccgcgc acatttcccc
     6361 gaaaagtgcc acctgac
//
