LOCUS       sequence-387244-        6488 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression of SpyCatcher-ELP-CarHCC-ELP-SpyCatcher, with TEV
            cleavage site and MMP cleavage site respectively before and after 
            each SpyCatcher, and GRGD site linked with (GGGGS)2 before CarHCC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6488)
  TITLE     Sun lab protein expression plasmids
REFERENCE   2  (bases 1 to 6488)
  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"
COMMENT     Sequence Label: pQE80l B-CC-B
FEATURES             Location/Qualifiers
     source          1..6488
                     /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             1561..1581
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             1588..1926
                     /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      2017..2111
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             2155..2814
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(2221..2240)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2879..2965
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3058..4140)
                     /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     4102..4121
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(4141..4218)
                     /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(4302..4324)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    4410..4550
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     4465..4484
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4565..4582)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4736..5324)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4816..4835)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5495..6286,6287..6355))
                     /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     6118..6137
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6356..6460)
                     /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 tagtccgccg ggtccgccgg tcctggtgac tacgccgccg
      961 ggcgaacgcc acgaaatcgg tgcgatgctg gcggcgtacc atctgcgtcg taagggcgtc
     1021 ccggcgctgt atctgggccc ggatactccg ctgccggacc tgcgtgcact ggcgcgccgc
     1081 ctgggtgcag gcgcggtcgt gctgtctgct gttctgagcg aaccgctgcg tgctctgcct
     1141 gacggtgccc tgaaagatct ggcaccgcgt gttttcctgg gcggccaggg cgcaggcccg
     1201 gaagaggcac gccgtctggg tgccgaatac atggaagacc tgaaaggcct ggctgaagcg
     1261 ctgtggctgc cgcgcggtcc ggaaaaagaa gcaatcacta gtgtgccggg cgtcggcgtg
     1321 ccgggcgtag gtgttccggg cgagggtgtt ccgggcgttg gtgtgccggg cgtcggcgtg
     1381 ccgggcgtgg gtgttccggg cgtaggtgtg ccgggcgagg gtgtgccggg cgtgggcgtg
     1441 ccgggcgtag gtgttccggg cgtaggtgtt ccgggcgtag gtgttccggg tgaaggcgtg
     1501 ccgggcgttg gtgtgccggg tgtgggcgtg ccgggcgggc tgctcgacat cccaacgacc
     1561 gaaaacctgt attttcaggg cgccatggtt gataccttat caggtttatc aagtgagcaa
     1621 ggtcagtccg gtgatatgac aattgaagaa gatagtgcta cccatattaa attctcaaaa
     1681 cgtgatgagg acggcaaaga gttagctggt gcaactatgg agttgcgtga ttcatctggt
     1741 aaaactatta gtacatggat ttcagatgga caagtgaaag atttctacct gtatccagga
     1801 aaatatacat ttgtcgaaac cgcagcacca gacggttatg aggtagcaac tgctattacc
     1861 tttacagtta atgagcaagg tcaggttact gtaaatggca aagcaactaa aggtgacgct
     1921 catattgacg gtccgcaagg tatttggggt cagctcgagt ggaagaaata aggatccggc
     1981 tgctaacaaa gcccgaagct taattagctg agcttggact cctgttgata gatccagtaa
     2041 tgacctcaga actccatctg gatttgttca gaacgctcgg ttgccgccgg gcgtttttta
     2101 ttggtgagaa tccaagccag cttggcgaga ttttcaggag ctaaggaagc taaaatggag
     2161 aaaaaaatca ctggatatac caccgttgat atatcccaat ggcatcgtaa agaacatttt
     2221 gaggcatttc agtcagttgc tcaatgtacc tataaccaga ccgttcagct ggatattacg
     2281 gcctttttaa agaccgtaaa gaaaaataag cacaagtttt atccggcctt tattcacatt
     2341 cttgcccgcc tgatgaatgc tcatccggaa tttcgtatgg caatgaaaga cggtgagctg
     2401 gtgatatggg atagtgttca cccttgttac accgttttcc atgagcaaac tgaaacgttt
     2461 tcatcgctct ggagtgaata ccacgacgat ttccggcagt ttctacacat atattcgcaa
     2521 gatgtggcgt gttacggtga aaacctggcc tatttcccta aagggtttat tgagaatatg
     2581 tttttcgtct cagccaatcc ctgggtgagt ttcaccagtt ttgatttaaa cgtggccaat
     2641 atggacaact tcttcgcccc cgttttcacc atgggcaaat attatacgca aggcgacaag
     2701 gtgctgatgc cgctggcgat tcaggttcat catgccgttt gtgatggctt ccatgtcggc
     2761 agaatgctta atgaattaca acagtactgc gatgagtggc agggcggggc gtaatttttt
     2821 taaggcagtt attggtgccc ttaaacgcct ggggtaatga ctctctagct tgaggcatca
     2881 aataaaacga aaggctcagt cgaaagactg ggcctttcgt tttatctgtt gtttgtcggt
     2941 gaacgctctc ctgagtagga caaatccgcc ctctagatta cgtgcagtcg atgataagct
     3001 gtcaaacatg agaattgtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     3061 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     3121 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     3181 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     3241 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     3301 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     3361 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     3421 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     3481 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     3541 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     3601 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     3661 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     3721 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     3781 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     3841 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     3901 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     3961 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     4021 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     4081 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     4141 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     4201 gcaccattcg atggtgtcgg aatttcgggc agcgttgggt cctggccacg ggtgcgcatg
     4261 atctagagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca catgcagctc
     4321 ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc ccgtcagggc
     4381 gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg tagcgatagc
     4441 ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga gtgcaccata
     4501 tgcggtgtga aataccgcac agatgcgtaa ggagaaaata ccgcatcagg cgctcttccg
     4561 cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc
     4621 actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt
     4681 gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc
     4741 ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa
     4801 acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc
     4861 ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg
     4921 cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc
     4981 tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc
     5041 gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca
     5101 ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact
     5161 acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca gttaccttcg
     5221 gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt
     5281 ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct
     5341 tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga
     5401 gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa
     5461 tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac
     5521 ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga
     5581 taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc
     5641 cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca
     5701 gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta
     5761 gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg
     5821 tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc
     5881 gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg
     5941 ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt
     6001 ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt
     6061 cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata
     6121 ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc
     6181 gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac
     6241 ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa
     6301 ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct
     6361 tcctttttca atattattga agcatttatc agggttattg tctcatgagc ggatacatat
     6421 ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc
     6481 cacctgac
//
