LOCUS       sequence-367536-        6084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes enzyme bovine carbonic anhydrase 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 6084)
  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 6084)
  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-BCA-P(11)4
FEATURES             Location/Qualifiers
     source          1..6084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     946..965
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(985..1062)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1268..1287
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1371..1390
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1371..1389
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1390..1414
                     /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             1429..1451
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2319..2336
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2389..2407)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2403..2450
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2487..2942
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2574..2593)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2784..2805
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3034..3849)
                     /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(3034..3849)
                     /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     3757..3776
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3841..3860
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3971..4559
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4460..4479
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4713..4730
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4745..4884
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4810..4829)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4970..4992
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4986..5177)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5986..6084,1..984))
                     /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"
ORIGIN
        1 ggtttttctt ttcaccagtg agacgggcaa cagctgattg cccttcaccg cctggccctg
       61 agagagttgc agcaagcggt ccacgctggt ttgccccagc aggcgaaaat cctgtttgat
      121 ggtggttaac ggcgggatat aacatgagct gtcttcggta tcgtcgtatc ccactaccga
      181 gatatccgca ccaacgcgca gcccggactc ggtaatggcg cgcattgcgc ccagcgccat
      241 ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc tcattcagca tttgcatggt
      301 ttgttgaaaa ccggacatgg cactccagtc gccttcccgt tccgctatcg gctgaatttg
      361 attgcgagtg agatatttat gccagccagc cagacgcaga cgcgccgaga cagaacttaa
      421 tgggcccgct aacagcgcga tttgctggtg acccaatgcg accagatgct ccacgcccag
      481 tcgcgtaccg tcttcatggg agaaaataat actgttgatg ggtgtctggt cagagacatc
      541 aagaaataac gccggaacat tagtgcaggc agcttccaca gcaatggcat cctggtcatc
      601 cagcggatag ttaatgatca gcccactgac gcgttgcgcg agaagattgt gcaccgccgc
      661 tttacaggct tcgacgccgc ttcgttctac catcgacacc accacgctgg cacccagttg
      721 atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc gcgtgcaggg ccagactgga
      781 ggtggcaacg ccaatcagca acgactgttt gcccgccagt tgttgtgcca cgcggttggg
      841 aatgtaattc agctccgcca tcgccgcttc cactttttcc cgcgttttcg cagaaacgtg
      901 gctggcctgg ttcaccacgc gggaaacggt ctgataagag acaccggcat actctgcgac
      961 atcgtataac gttactggtt tcacattcac caccctgaat tgactctctt ccgggcgcta
     1021 tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg tccgggatct cgacgctctc
     1081 ccttatgcga ctcctgcatt aggaagcagc ccagtagtag gttgaggccg ttgagcaccg
     1141 ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa cagtcccccg gccacggggc
     1201 ctgccaccat acccacgccg aaacaagcgc tcatgagccc gaagtggcga gcccgatctt
     1261 ccccatcggt gatgtcggcg atataggcgc cagcaaccgc acctgtggcg ccggtgatgc
     1321 cggccacgat gcgtccggcg tagaggatcg agatctcgat cccgcgaaat taatacgact
     1381 cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt tgtttaactt
     1441 taagaaggag atataccatg ggtatgtcac accattgggg ctacggtaaa cacaacggtc
     1501 cggaacactg gcacaaagac ttcccgattg ctaacggcga acgtcagtca ccggtcgata
     1561 ttgacaccaa agcagtggtt caggatccgg cactgaaacc gctggctctg gtttatggcg
     1621 aagctacgtc gcgtcgcatg gtcaacaatg gtcatagctt taacgtggaa tatgatgact
     1681 ctcaggataa agcagtgctg aaagacggcc cgctgaccgg cacgtaccgt ctggttcagt
     1741 ttcatttcca ctggggcagc tctgatgacc aaggttcaga acacaccgtt gatcgcaaaa
     1801 aatatgcggc cgaactgcat ctggtccact ggaacaccaa atacggcgat tttggtaccg
     1861 cagcgcagca accggatggt ctggccgttg tgggtgtttt cctgaaagtc ggcgatgcga
     1921 atccggccct gcagaaagtg ctggatgccc tggactcaat caaaaccaaa ggcaaatcga
     1981 cggattttcc gaacttcgac ccgggtagcc tgctgccgaa tgttctggat tattggacct
     2041 atccgggtag tctgaccacg ccgccgctgc tggaatccgt gacgtggatt gttctgaaag
     2101 aaccgatctc tgtgagttcc cagcaaatgc tgaaatttcg taccctgaac ttcaatgccg
     2161 aaggtgaacc ggaactgctg atgctggcca actggcgtcc ggcacagccg ctgaaaaatc
     2221 gtcaagtgcg cggcttcccg aaaggtggtg gtggtagtgg cggcggtggc agtcagcaac
     2281 gctttgaatg ggaatttgaa caacaataat aactcgagca ccaccaccac caccactgag
     2341 atccggctgc taacaaagcc cgaaaggaag ctgagttggc tgctgccacc gctgagcaat
     2401 aactagcata accccttggg gcctctaaac gggtcttgag gggttttttg ctgaaaggag
     2461 gaactatatc cggattggcg aatgggacgc gccctgtagc ggcgcattaa gcgcggcggg
     2521 tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt
     2581 cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg
     2641 ggggctccct ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga
     2701 ttagggtgat ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac
     2761 gttggagtcc acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc
     2821 tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct attggttaaa
     2881 aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa cgcttacaat
     2941 ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     3001 cattcaaata tgtatccgct catgaattaa ttcttagaaa aactcatcga gcatcaaatg
     3061 aaactgcaat ttattcatat caggattatc aataccatat ttttgaaaaa gccgtttctg
     3121 taatgaagga gaaaactcac cgaggcagtt ccataggatg gcaagatcct ggtatcggtc
     3181 tgcgattccg actcgtccaa catcaataca acctattaat ttcccctcgt caaaaataag
     3241 gttatcaagt gagaaatcac catgagtgac gactgaatcc ggtgagaatg gcaaaagttt
     3301 atgcatttct ttccagactt gttcaacagg ccagccatta cgctcgtcat caaaatcact
     3361 cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga gcgagacgaa atacgcgatc
     3421 gctgttaaaa ggacaattac aaacaggaat cgaatgcaac cggcgcagga acactgccag
     3481 cgcatcaaca atattttcac ctgaatcagg atattcttct aatacctgga atgctgtttt
     3541 cccggggatc gcagtggtga gtaaccatgc atcatcagga gtacggataa aatgcttgat
     3601 ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg accatctcat ctgtaacatc
     3661 attggcaacg ctacctttgc catgtttcag aaacaactct ggcgcatcgg gcttcccata
     3721 caatcgatag attgtcgcac ctgattgccc gacattatcg cgagcccatt tatacccata
     3781 taaatcagca tccatgttgg aatttaatcg cggcctagag caagacgttt cccgttgaat
     3841 atggctcata acaccccttg tattactgtt tatgtaagca gacagtttta ttgttcatga
     3901 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     3961 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     4021 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     4081 taactggctt cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag
     4141 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     4201 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     4261 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     4321 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     4381 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     4441 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     4501 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     4561 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     4621 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     4681 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag
     4741 agcgcctgat gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg
     4801 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg
     4861 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga
     4921 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc
     4981 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca
     5041 tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg
     5101 agtttctcca gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt
     5161 ttttcctgtt tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg
     5221 ataccgatga aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg
     5281 ttactggaac gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa
     5341 atcactcagg gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc
     5401 cagcagcatc ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt
     5461 tccagacttt acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac
     5521 gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca
     5581 gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc
     5641 cgtggggccg ccatgccggc gataatggcc tgcttctcgc cgaaacgttt ggtggcggga
     5701 ccagtgacga aggcttgagc gagggcgtgc aagattccga ataccgcaag cgacaggccg
     5761 atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa tgacccagag cgctgccggc
     5821 acctgtccta cgagttgcat gataaagaag acagtcataa gtgcggcgac gatagtcatg
     5881 ccccgcgccc accggaagga gctgactggg ttgaaggctc tcaagggcat cggtcgagat
     5941 cccggtgcct aatgagtgag ctaacttaca ttaattgcgt tgcgctcact gcccgctttc
     6001 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     6061 ggtttgcgta ttgggcgcca gggt
//
