LOCUS       sequence-367541-        6003 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes enzyme cutinase linked to P(11)4 self-assembling peptide via
            a GS-linker sequence.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6003)
  AUTHORS   Shanbhag BK, Liu C, Pradeep GC, Younas T, Hu KKY, Fulcher AJ, Struwe
            WB, Steer D, Dumsday G, Harper IS, Kukura P, Haritos VS, He L
  TITLE     Custom Design of Protein Particles as Multifunctional Biomaterials
  JOURNAL   Adv. Funct. Mater. 2022, 32, 2108039
REFERENCE   2  (bases 1 to 6003)
  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: "Adv. Funct.
            Mater. 2022, 32, 2108039"
COMMENT     Sequence Label: pET28a-Cut-P(11)4
FEATURES             Location/Qualifiers
     source          1..6003
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..46
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     130..149
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        130..148
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    149..173
                     /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             188..210
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             229..246
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             256..273
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(534..551)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             997..1014
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1067..1085)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1081..1128
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1165..1620
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1252..1271)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1462..1483
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1712..2527)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(1712..2527)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     2435..2454
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2519..2538
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2649..3237
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3138..3157
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3391..3408
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3423..3562
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3488..3507)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3648..3670
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3664..3855)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4664..5746)
                     /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     5708..5727
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5747..5824)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 aagtggcgag cccgatcttc cccatcggtg atgtcggcga tataggcgcc agcaaccgca
       61 cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt agaggatcga gatctcgatc
      121 ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag
      181 aaataatttt gtttaacttt aagaaggaga tataccatgg gcagcagcca tcatcatcat
      241 catcacagca gcggcctggt gccgcgcggc agccatatgc tgccgaccag caacccggcg
      301 caggagctgg aagcgcgtca actgggccgt accacccgtg acgatctgat caacggtaac
      361 agcgcgagct gcgcggacgt gatcttcatt tacgcgcgtg gcagcaccga aaccggtaac
      421 ctgggcaccc tgggtccgag cattgcgagc aacctggaga gcgcgtttgg caaggacggt
      481 gtgtggattc agggtgttgg tggcgcgtat cgtgcgaccc tgggtgataa cgcgctgccg
      541 cgtggcacca gcagcgcggc gatccgtgaa atgctgggcc tgttccagca agcgaacacc
      601 aaatgcccgg atgcgaccct gattgcgggt ggctacagcc agggtgcggc gctggcggcg
      661 gcgagcattg aggacctgga tagcgcgatc cgtgataaga ttgcgggcac cgtgctgttc
      721 ggttacacca aaaacctgca aaaccgtggt cgtattccga actatccggc ggaccgtacc
      781 aaggtgttct gcaacaccgg cgatctggtg tgcaccggta gcctgattgt tgcggcgccg
      841 cacctggcgt atggcccgga tgcgcgtggt ccggcgccgg aatttctgat tgagaaagtg
      901 cgtgcggttc gtggtagcgc gggtggcggt ggcagcggtg gcggtggcag ccagcaacgt
      961 ttcgagtggg aatttgagca gcaataataa ctcgagcacc accaccacca ccactgagat
     1021 ccggctgcta acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     1081 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     1141 actatatccg gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     1201 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     1261 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     1321 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     1381 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     1441 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     1501 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     1561 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     1621 aggtggcact tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca
     1681 ttcaaatatg tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa
     1741 actgcaattt attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta
     1801 atgaaggaga aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg
     1861 cgattccgac tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt
     1921 tatcaagtga gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat
     1981 gcatttcttt ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg
     2041 catcaaccaa accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc
     2101 tgttaaaagg acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg
     2161 catcaacaat attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc
     2221 cggggatcgc agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg
     2281 tcggaagagg cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat
     2341 tggcaacgct acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca
     2401 atcgatagat tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata
     2461 aatcagcatc catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat
     2521 ggctcataac accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc
     2581 aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa
     2641 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
     2701 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
     2761 actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc
     2821 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
     2881 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
     2941 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
     3001 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
     3061 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
     3121 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
     3181 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
     3241 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
     3301 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat
     3361 accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag
     3421 cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca
     3481 ctctcagtac aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct
     3541 acgtgactgg gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg
     3601 ggcttgtctg ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat
     3661 gtgtcagagg ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc
     3721 agcgtggtcg tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag
     3781 tttctccaga agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt
     3841 ttcctgtttg gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat
     3901 accgatgaaa cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt
     3961 actggaacgt tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat
     4021 cactcagggt caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca
     4081 gcagcatcct gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc
     4141 cagactttac gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt
     4201 tttgcagcag cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt
     4261 aaggcaaccc cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg
     4321 tggggccgcc atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc
     4381 agtgacgaag gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat
     4441 catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac
     4501 ctgtcctacg agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc
     4561 ccgcgcccac cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc
     4621 cggtgcctaa tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca
     4681 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     4741 tttgcgtatt gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat
     4801 tgcccttcac cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca
     4861 gcaggcgaaa atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg
     4921 tatcgtcgta tcccactacc gagatatccg caccaacgcg cagcccggac tcggtaatgg
     4981 cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc
     5041 cctcattcag catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc
     5101 gttccgctat cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca
     5161 gacgcgccga gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg
     5221 cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga
     5281 tgggtgtctg gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca
     5341 cagcaatggc atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg
     5401 cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca
     5461 ccaccacgct ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg
     5521 gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca
     5581 gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt
     5641 cccgcgtttt cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag
     5701 agacaccggc atactctgcg acatcgtata acgttactgg tttcacattc accaccctga
     5761 attgactctc ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg
     5821 tgtccgggat ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt
     5881 aggttgaggc cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc
     5941 aacagtcccc cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc
     6001 ccg
//
