LOCUS       sequence-221418-        5393 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Two SpyTags linked by an elastin-like polypeptide for hydrogel 
            formation.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5393)
  AUTHORS   Wieduwild R, Howarth M
  TITLE     Assembling and decorating hyaluronan hydrogels with twin protein 
            superglues to mimic cell-cell interactions.
  JOURNAL   Biomaterials. 2018 Oct;180:253-264. doi: 
            10.1016/j.biomaterials.2018.07.020. Epub 2018 Jul 17.
  PUBMED    30053659
REFERENCE   2  (bases 1 to 5393)
  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: 
            "Biomaterials."; date: "2018-10"; volume: "180:253-264. doi"; pages:
            " 10.1016/j.biomaterials.2018.07.020. Epub 2018 Jul 17"
COMMENT     Sequence Label: pQE80L SpyTag-ELP-SpyTag
FEATURES             Location/Qualifiers
     source          1..5393
                     /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             820..858
                     /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      922..1016
                     /label=lambda t0 terminator
                     /note="transcription terminator from phage lambda"
     CDS             1060..1719
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(1126..1145)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     terminator      1784..1870
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(1963..3045)
                     /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     3007..3026
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(3046..3123)
                     /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(3207..3229)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    3315..3455
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     3370..3389
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(3470..3487)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3641..4229)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3721..3740)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4400..5191,5192..5260))
                     /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     5023..5042
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5261..5365)
                     /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 gagctctatg cggttaccgg ccgtggtcgt
      541 gccccggcct gctctgcccc gatcgccact agtgtgccgg gcgtcggcgt gccgggcgta
      601 ggtgttccgg gcgagggtgt tccgggcgtt ggtgtgccgg gcgtcggcgt gccgggcgtg
      661 ggtgttccgg gcgtaggtgt gccgggcgag ggtgtgccgg gcgtgggcgt gccgggcgta
      721 ggtgttccgg gcgtaggtgt tccgggcgta ggtgttccgg gtgaaggcgt gccgggcgtt
      781 ggtgtgccgg gtgtgggcgt gccgggcggg ctgctcgacg cccatattgt catggttgat
      841 gcatacaagc cgacgaagct cgagtggaag aaataaggat ccggctgcta acaaagcccg
      901 aagcttaatt agctgagctt ggactcctgt tgatagatcc agtaatgacc tcagaactcc
      961 atctggattt gttcagaacg ctcggttgcc gccgggcgtt ttttattggt gagaatccaa
     1021 gccagcttgg cgagattttc aggagctaag gaagctaaaa tggagaaaaa aatcactgga
     1081 tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc atttcagtca
     1141 gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt tttaaagacc
     1201 gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc ccgcctgatg
     1261 aatgctcatc cggaatttcg tatggcaatg aaagacggtg agctggtgat atgggatagt
     1321 gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc gctctggagt
     1381 gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt ggcgtgttac
     1441 ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt cgtctcagcc
     1501 aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga caacttcttc
     1561 gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct gatgccgctg
     1621 gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat gcttaatgaa
     1681 ttacaacagt actgcgatga gtggcagggc ggggcgtaat ttttttaagg cagttattgg
     1741 tgcccttaaa cgcctggggt aatgactctc tagcttgagg catcaaataa aacgaaaggc
     1801 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
     1861 taggacaaat ccgccctcta gattacgtgc agtcgatgat aagctgtcaa acatgagaat
     1921 tgtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag
     1981 tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt
     2041 ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt
     2101 gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag
     2161 caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt
     2221 atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc
     2281 gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc
     2341 ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg
     2401 ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag
     2461 acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc
     2521 gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat
     2581 gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac
     2641 agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc
     2701 gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac
     2761 caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg
     2821 cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag
     2881 ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc
     2941 ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga
     3001 gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa
     3061 ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcacc attcgatggt
     3121 gtcggaattt cgggcagcgt tgggtcctgg ccacgggtgc gcatgatcta gagctgcctc
     3181 gcgcgtttcg gtgatgacgg tgaaaacctc tgacacatgc agctcccgga gacggtcaca
     3241 gcttgtctgt aagcggatgc cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt
     3301 ggcgggtgtc ggggcgcagc catgacccag tcacgtagcg atagcggagt gtatactggc
     3361 ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg tgtgaaatac
     3421 cgcacagatg cgtaaggaga aaataccgca tcaggcgctc ttccgcttcc tcgctcactg
     3481 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     3541 tacggttatc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     3601 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     3661 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     3721 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     3781 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     3841 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     3901 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     3961 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     4021 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     4081 ggacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     4141 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     4201 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     4261 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     4321 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     4381 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     4441 gtctatttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     4501 agggcttacc atctggcccc agtgctgcaa tgataccgcg agacccacgc tcaccggctc
     4561 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     4621 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     4681 cagttaatag tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt
     4741 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     4801 ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     4861 tggccgcagt gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc
     4921 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     4981 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     5041 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     5101 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     5161 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     5221 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     5281 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     5341 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gac
//
