LOCUS       sequence-387269-        5756 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression of SpyTag-ELP-CarHCC-ELP SpyTag, with GRGD site 
            linked with (GGGGS)2 before CarHCC and after first ELP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5756)
  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 5756)
  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 A-CC-A
FEATURES             Location/Qualifiers
     source          1..5756
                     /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             226..264
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     CDS             1183..1221
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     terminator      1285..1379
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1423..2082
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1489..1508)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2147..2233
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2326..3408)
                     /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     3370..3389
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3409..3486)
                     /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(3570..3592)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3678..3818
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3733..3752
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3833..3850)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4004..4592)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4084..4103)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4763..5554,5555..5623))
                     /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     5386..5405
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5624..5728)
                     /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 tcgacgccca tattgtcatg
      241 gttgatgcat acaagccgac gaagctcgac ggccacggcg tgggtgttcc gggcgtcggt
      301 gtgccgggtg tgggtgtgcc gggcgaaggt gtgccgggcg tcggtgtgcc gggtgttggt
      361 gttccgggcg ttggtgtgcc gggcgttggc gtgccgggcg agggtgtgcc gggcgttggt
      421 gttccgggcg tgggtgtgcc gggcgtgggc gtgccgggcg tcggtgttcc gggcgagggt
      481 gtgccgggcg taggtgtgcc gggcgttggt gagctcggcc gtggtgatgg tggtgggggt
      541 agcggtggtg ggggtagtcc gccgggtccg ccggtcctgg tgactacgcc gccgggcgaa
      601 cgccacgaaa tcggtgcgat gctggcggcg taccatctgc gtcgtaaggg cgtcccggcg
      661 ctgtatctgg gcccggatac tccgctgccg gacctgcgtg cactggcgcg ccgcctgggt
      721 gcaggcgcgg tcgtgctgtc tgctgttctg agcgaaccgc tgcgtgctct gcctgacggt
      781 gccctgaaag atctggcacc gcgtgttttc ctgggcggcc agggcgcagg cccggaagag
      841 gcacgccgtc tgggtgccga atacatggaa gacctgaaag gcctggctga agcgctgtgg
      901 ctgccgcgcg gtccggaaaa agaagcaatc actagtgtgc cgggcgtcgg cgtgccgggc
      961 gtaggtgttc cgggcgaggg tgttccgggc gttggtgtgc cgggcgtcgg cgtgccgggc
     1021 gtgggtgttc cgggcgtagg tgtgccgggc gagggtgtgc cgggcgtggg cgtgccgggc
     1081 gtaggtgttc cgggcgtagg tgttccgggc gtaggtgttc cgggtgaagg cgtgccgggc
     1141 gttggtgtgc cgggtgtggg cgtgccgggc gggctgctcg acgcccatat tgtcatggtt
     1201 gatgcataca agccgacgaa gctcgagtgg aagaaataag gatccggctg ctaacaaagc
     1261 ccgaagctta attagctgag cttggactcc tgttgataga tccagtaatg acctcagaac
     1321 tccatctgga tttgttcaga acgctcggtt gccgccgggc gttttttatt ggtgagaatc
     1381 caagccagct tggcgagatt ttcaggagct aaggaagcta aaatggagaa aaaaatcact
     1441 ggatatacca ccgttgatat atcccaatgg catcgtaaag aacattttga ggcatttcag
     1501 tcagttgctc aatgtaccta taaccagacc gttcagctgg atattacggc ctttttaaag
     1561 accgtaaaga aaaataagca caagttttat ccggccttta ttcacattct tgcccgcctg
     1621 atgaatgctc atccggaatt tcgtatggca atgaaagacg gtgagctggt gatatgggat
     1681 agtgttcacc cttgttacac cgttttccat gagcaaactg aaacgttttc atcgctctgg
     1741 agtgaatacc acgacgattt ccggcagttt ctacacatat attcgcaaga tgtggcgtgt
     1801 tacggtgaaa acctggccta tttccctaaa gggtttattg agaatatgtt tttcgtctca
     1861 gccaatccct gggtgagttt caccagtttt gatttaaacg tggccaatat ggacaacttc
     1921 ttcgcccccg ttttcaccat gggcaaatat tatacgcaag gcgacaaggt gctgatgccg
     1981 ctggcgattc aggttcatca tgccgtttgt gatggcttcc atgtcggcag aatgcttaat
     2041 gaattacaac agtactgcga tgagtggcag ggcggggcgt aattttttta aggcagttat
     2101 tggtgccctt aaacgcctgg ggtaatgact ctctagcttg aggcatcaaa taaaacgaaa
     2161 ggctcagtcg aaagactggg cctttcgttt tatctgttgt ttgtcggtga acgctctcct
     2221 gagtaggaca aatccgccct ctagattacg tgcagtcgat gataagctgt caaacatgag
     2281 aattgtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     2341 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     2401 ggtttgcgta ttgggcgcca gggtggtttt tcttttcacc agtgagacgg gcaacagctg
     2461 attgcccttc accgcctggc cctgagagag ttgcagcaag cggtccacgc tggtttgccc
     2521 cagcaggcga aaatcctgtt tgatggtggt taacggcggg atataacatg agctgtcttc
     2581 ggtatcgtcg tatcccacta ccgagatatc cgcaccaacg cgcagcccgg actcggtaat
     2641 ggcgcgcatt gcgcccagcg ccatctgatc gttggcaacc agcatcgcag tgggaacgat
     2701 gccctcattc agcatttgca tggtttgttg aaaaccggac atggcactcc agtcgccttc
     2761 ccgttccgct atcggctgaa tttgattgcg agtgagatat ttatgccagc cagccagacg
     2821 cagacgcgcc gagacagaac ttaatgggcc cgctaacagc gcgatttgct ggtgacccaa
     2881 tgcgaccaga tgctccacgc ccagtcgcgt accgtcttca tgggagaaaa taatactgtt
     2941 gatgggtgtc tggtcagaga catcaagaaa taacgccgga acattagtgc aggcagcttc
     3001 cacagcaatg gcatcctggt catccagcgg atagttaatg atcagcccac tgacgcgttg
     3061 cgcgagaaga ttgtgcaccg ccgctttaca ggcttcgacg ccgcttcgtt ctaccatcga
     3121 caccaccacg ctggcaccca gttgatcggc gcgagattta atcgccgcga caatttgcga
     3181 cggcgcgtgc agggccagac tggaggtggc aacgccaatc agcaacgact gtttgcccgc
     3241 cagttgttgt gccacgcggt tgggaatgta attcagctcc gccatcgccg cttccacttt
     3301 ttcccgcgtt ttcgcagaaa cgtggctggc ctggttcacc acgcgggaaa cggtctgata
     3361 agagacaccg gcatactctg cgacatcgta taacgttact ggtttcacat tcaccaccct
     3421 gaattgactc tcttccgggc gctatcatgc cataccgcga aaggttttgc accattcgat
     3481 ggtgtcggaa tttcgggcag cgttgggtcc tggccacggg tgcgcatgat ctagagctgc
     3541 ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
     3601 acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt
     3661 gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact
     3721 ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa
     3781 taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca
     3841 ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg
     3901 taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc
     3961 agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc
     4021 cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac
     4081 tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc
     4141 tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata
     4201 gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc
     4261 acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca
     4321 acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag
     4381 cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta
     4441 gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg
     4501 gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc
     4561 agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt
     4621 ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa
     4681 ggatcttcac ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat
     4741 atgagtaaac ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga
     4801 tctgtctatt tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac
     4861 gggagggctt accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg
     4921 ctccagattt atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg
     4981 caactttatc cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt
     5041 cgccagttaa tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct
     5101 cgtcgtttgg tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat
     5161 cccccatgtt gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta
     5221 agttggccgc agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca
     5281 tgccatccgt aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat
     5341 agtgtatgcg gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac
     5401 atagcagaac tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa
     5461 ggatcttacc gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt
     5521 cagcatcttt tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg
     5581 caaaaaaggg aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat
     5641 attattgaag catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt
     5701 agaaaaataa acaaataggg gttccgcgca catttccccg aaaagtgcca cctgac
//
