LOCUS       sequence-387232-        5960 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression of SpyTag-ELP-CarHC-ELP-SpyTag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5960)
  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 5960)
  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 ACA
FEATURES             Location/Qualifiers
     source          1..5960
                     /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             1387..1425
                     /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      1489..1583
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1627..2286
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1693..1712)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      2351..2437
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(2530..3612)
                     /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     3574..3593
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3613..3690)
                     /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(3774..3796)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3882..4022
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3937..3956
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(4037..4054)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4208..4796)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4288..4307)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4967..5758,5759..5827))
                     /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     5590..5609
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5828..5932)
                     /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 gagctcccag aagatctggg caccggcctg
      541 ctggaagcac tgctgcgcgg tgatctggcg ggcgccgaag ctctgtttcg tcgtggcctg
      601 cgtttctggg gcccggaagg cgttctggag cacctgctgc tgccggtgct gcgtgaagtg
      661 ggcgaagctt ggcaccgtgg tgaaatcggc gttgcagaag aacacctggc gagcaccttc
      721 ctgcgcgctc gtctgcagga gctgctggac ctggcaggtt tcccgccggg tccgccggtc
      781 ctggtgacta cgccaccggg cgaacgccac gaaatcggtg cgatgctggc ggcgtaccat
      841 ctgcgtcgta agggcgtccc ggcgctgtat ctgggcccgg atactccgct gccggacctg
      901 cgtgcactgg cgcgccgcct gggtgcaggc gcggtcgtgc tgtctgctgt tctgagcgaa
      961 ccgctgcgtg ctctgcctga cggtgccctg aaagatctgg caccgcgtgt tttcctgggc
     1021 ggccagggcg caggcccgga agaggcacgc cgtctgggtg ccgaatacat ggaagacctg
     1081 aaaggcctgg ctgaagcgct gtggctgccg cgcggtccgg aaaaagaagc aatcactagt
     1141 gtgccgggcg tcggcgtgcc gggcgtaggt gttccgggcg agggtgttcc gggcgttggt
     1201 gtgccgggcg tcggcgtgcc gggcgtgggt gttccgggcg taggtgtgcc gggcgagggt
     1261 gtgccgggcg tgggcgtgcc gggcgtaggt gttccgggcg taggtgttcc gggcgtaggt
     1321 gttccgggtg aaggcgtgcc gggcgttggt gtgccgggtg tgggcgtgcc gggcgggctg
     1381 ctcgacgccc atattgtcat ggttgatgca tacaagccga cgaagctcga gtggaagaaa
     1441 taaggatccg gctgctaaca aagcccgaag cttaattagc tgagcttgga ctcctgttga
     1501 tagatccagt aatgacctca gaactccatc tggatttgtt cagaacgctc ggttgccgcc
     1561 gggcgttttt tattggtgag aatccaagcc agcttggcga gattttcagg agctaaggaa
     1621 gctaaaatgg agaaaaaaat cactggatat accaccgttg atatatccca atggcatcgt
     1681 aaagaacatt ttgaggcatt tcagtcagtt gctcaatgta cctataacca gaccgttcag
     1741 ctggatatta cggccttttt aaagaccgta aagaaaaata agcacaagtt ttatccggcc
     1801 tttattcaca ttcttgcccg cctgatgaat gctcatccgg aatttcgtat ggcaatgaaa
     1861 gacggtgagc tggtgatatg ggatagtgtt cacccttgtt acaccgtttt ccatgagcaa
     1921 actgaaacgt tttcatcgct ctggagtgaa taccacgacg atttccggca gtttctacac
     1981 atatattcgc aagatgtggc gtgttacggt gaaaacctgg cctatttccc taaagggttt
     2041 attgagaata tgtttttcgt ctcagccaat ccctgggtga gtttcaccag ttttgattta
     2101 aacgtggcca atatggacaa cttcttcgcc cccgttttca ccatgggcaa atattatacg
     2161 caaggcgaca aggtgctgat gccgctggcg attcaggttc atcatgccgt ttgtgatggc
     2221 ttccatgtcg gcagaatgct taatgaatta caacagtact gcgatgagtg gcagggcggg
     2281 gcgtaatttt tttaaggcag ttattggtgc ccttaaacgc ctggggtaat gactctctag
     2341 cttgaggcat caaataaaac gaaaggctca gtcgaaagac tgggcctttc gttttatctg
     2401 ttgtttgtcg gtgaacgctc tcctgagtag gacaaatccg ccctctagat tacgtgcagt
     2461 cgatgataag ctgtcaaaca tgagaattgt gcctaatgag tgagctaact tacattaatt
     2521 gcgttgcgct cactgcccgc tttccagtcg ggaaacctgt cgtgccagct gcattaatga
     2581 atcggccaac gcgcggggag aggcggtttg cgtattgggc gccagggtgg tttttctttt
     2641 caccagtgag acgggcaaca gctgattgcc cttcaccgcc tggccctgag agagttgcag
     2701 caagcggtcc acgctggttt gccccagcag gcgaaaatcc tgtttgatgg tggttaacgg
     2761 cgggatataa catgagctgt cttcggtatc gtcgtatccc actaccgaga tatccgcacc
     2821 aacgcgcagc ccggactcgg taatggcgcg cattgcgccc agcgccatct gatcgttggc
     2881 aaccagcatc gcagtgggaa cgatgccctc attcagcatt tgcatggttt gttgaaaacc
     2941 ggacatggca ctccagtcgc cttcccgttc cgctatcggc tgaatttgat tgcgagtgag
     3001 atatttatgc cagccagcca gacgcagacg cgccgagaca gaacttaatg ggcccgctaa
     3061 cagcgcgatt tgctggtgac ccaatgcgac cagatgctcc acgcccagtc gcgtaccgtc
     3121 ttcatgggag aaaataatac tgttgatggg tgtctggtca gagacatcaa gaaataacgc
     3181 cggaacatta gtgcaggcag cttccacagc aatggcatcc tggtcatcca gcggatagtt
     3241 aatgatcagc ccactgacgc gttgcgcgag aagattgtgc accgccgctt tacaggcttc
     3301 gacgccgctt cgttctacca tcgacaccac cacgctggca cccagttgat cggcgcgaga
     3361 tttaatcgcc gcgacaattt gcgacggcgc gtgcagggcc agactggagg tggcaacgcc
     3421 aatcagcaac gactgtttgc ccgccagttg ttgtgccacg cggttgggaa tgtaattcag
     3481 ctccgccatc gccgcttcca ctttttcccg cgttttcgca gaaacgtggc tggcctggtt
     3541 caccacgcgg gaaacggtct gataagagac accggcatac tctgcgacat cgtataacgt
     3601 tactggtttc acattcacca ccctgaattg actctcttcc gggcgctatc atgccatacc
     3661 gcgaaaggtt ttgcaccatt cgatggtgtc ggaatttcgg gcagcgttgg gtcctggcca
     3721 cgggtgcgca tgatctagag ctgcctcgcg cgtttcggtg atgacggtga aaacctctga
     3781 cacatgcagc tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa
     3841 gcccgtcagg gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca
     3901 cgtagcgata gcggagtgta tactggctta actatgcggc atcagagcag attgtactga
     3961 gagtgcacca tatgcggtgt gaaataccgc acagatgcgt aaggagaaaa taccgcatca
     4021 ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag
     4081 cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
     4141 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc
     4201 tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc
     4261 agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc
     4321 tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt
     4381 cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg
     4441 ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
     4501 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag
     4561 ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt
     4621 ggtggcctaa ctacggctac actagaagga cagtatttgg tatctgcgct ctgctgaagc
     4681 cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta
     4741 gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag
     4801 atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
     4861 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa
     4921 gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa
     4981 tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc
     5041 ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga
     5101 taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa
     5161 gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
     5221 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg
     5281 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc
     5341 aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg
     5401 gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag
     5461 cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt
     5521 actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
     5581 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac
     5641 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac
     5701 ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag
     5761 caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa
     5821 tactcatact cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga
     5881 gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
     5941 cccgaaaagt gccacctgac
//
