LOCUS       sequence-367540-        6249 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes thermostable carbonic anhydrase enzyme 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 6249)
  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 6249)
  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-TmCA-P(11)4
FEATURES             Location/Qualifiers
     source          1..6249
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     50..69
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     153..172
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        153..171
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    172..196
                     /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             211..233
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             252..269
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             279..296
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1266..1283
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(1336..1354)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1350..1397
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1434..1889
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1521..1540)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1731..1752
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1981..2796)
                     /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(1981..2796)
                     /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     2704..2723
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2788..2807
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2918..3506
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3407..3426
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3660..3677
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3692..3831
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3757..3776)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3917..3939
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3933..4124)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4933..6015)
                     /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     5977..5996
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6016..6093)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
       61 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
      121 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
      181 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca
      241 tgggcagcag ccatcatcat catcatcaca gcagcggcct ggtgccgcgc ggcagccata
      301 tggcaaataa cgtggccgca ccgctgatcg acctgggcgc agaagctaaa aaacaagcac
      361 agaaatccgc cgctacgcaa tccgcagtcc cggaaaaaga aagtgcaacc aaagtggctg
      421 aaaaacagaa agaaccggaa gaaaaagcaa aaccggaacc gaaaaaaccg ccgcattggg
      481 gctattttgg tgaagaaggc ccgcaatact ggggtgaact ggctccggaa ttcagtacct
      541 gcaaaacggg caaaaaccag tccccgatta atctgaaacc gcaaaccgcg gttggtacca
      601 cgtctctgcc gggctttgat gtctattacc gcgaaaccgc cctgaaactg attaacaatg
      661 gtcacacgct gcaggtgaac atcccgctgg gtagttatat taaaatcaat ggccatcgct
      721 atgaactgct gcagtaccat tttcacacgc cgtccgaaca ccaacgtgac ggtttcaact
      781 atccgatgga aatgcatctg gtgcacaaag atggcgacgg taacctggcg gttattgcca
      841 tcctgtttca ggaaggcgaa gaaaatgaaa ccctggcgaa actgatgagc ttcctgccgc
      901 agacgctgaa aaaacaagaa atccatgaaa gcgtgaaaat ccacccggcg aaatttttcc
      961 cggccgataa aaaattctac aaatacagcg gttctctgac cacgccgccg tgttcagaag
     1021 gcgtgtattg gatggttttc aaacagccga tccaagcgtc agtcacccag ctggaaaaaa
     1081 tgcatgaata cctgggttcg aacgcacgtc cggtgcagcg ccaaaatgct cgtaccctgc
     1141 tgaaatcgtg gccggaccgt aaccgcgcca atacggtcta tgaattctac ggcggcggtg
     1201 gcagcggcgg cggtggctca cagcagcgtt ttgaatggga atttgaacaa cagtaataac
     1261 tcgagcacca ccaccaccac cactgagatc cggctgctaa caaagcccga aaggaagctg
     1321 agttggctgc tgccaccgct gagcaataac tagcataacc ccttggggcc tctaaacggg
     1381 tcttgagggg ttttttgctg aaaggaggaa ctatatccgg attggcgaat gggacgcgcc
     1441 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     1501 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     1561 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     1621 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     1681 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     1741 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     1801 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     1861 ttttaacaaa atattaacgc ttacaattta ggtggcactt ttcggggaaa tgtgcgcgga
     1921 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gaattaattc
     1981 ttagaaaaac tcatcgagca tcaaatgaaa ctgcaattta ttcatatcag gattatcaat
     2041 accatatttt tgaaaaagcc gtttctgtaa tgaaggagaa aactcaccga ggcagttcca
     2101 taggatggca agatcctggt atcggtctgc gattccgact cgtccaacat caatacaacc
     2161 tattaatttc ccctcgtcaa aaataaggtt atcaagtgag aaatcaccat gagtgacgac
     2221 tgaatccggt gagaatggca aaagtttatg catttctttc cagacttgtt caacaggcca
     2281 gccattacgc tcgtcatcaa aatcactcgc atcaaccaaa ccgttattca ttcgtgattg
     2341 cgcctgagcg agacgaaata cgcgatcgct gttaaaagga caattacaaa caggaatcga
     2401 atgcaaccgg cgcaggaaca ctgccagcgc atcaacaata ttttcacctg aatcaggata
     2461 ttcttctaat acctggaatg ctgttttccc ggggatcgca gtggtgagta accatgcatc
     2521 atcaggagta cggataaaat gcttgatggt cggaagaggc ataaattccg tcagccagtt
     2581 tagtctgacc atctcatctg taacatcatt ggcaacgcta cctttgccat gtttcagaaa
     2641 caactctggc gcatcgggct tcccatacaa tcgatagatt gtcgcacctg attgcccgac
     2701 attatcgcga gcccatttat acccatataa atcagcatcc atgttggaat ttaatcgcgg
     2761 cctagagcaa gacgtttccc gttgaatatg gctcataaca ccccttgtat tactgtttat
     2821 gtaagcagac agttttattg ttcatgacca aaatccctta acgtgagttt tcgttccact
     2881 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     2941 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     3001 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     3061 ctgtccttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     3121 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     3181 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     3241 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     3301 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     3361 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     3421 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     3481 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     3541 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     3601 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     3661 gcgagtcagt gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc
     3721 tgtgcggtat ttcacaccgc aatggtgcac tctcagtaca atctgctctg atgccgcata
     3781 gttaagccag tatacactcc gctatcgcta cgtgactggg tcatggctgc gccccgacac
     3841 ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga
     3901 caagctgtga ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa
     3961 cgcgcgaggc agctgcggta aagctcatca gcgtggtcgt gaagcgattc acagatgtct
     4021 gcctgttcat ccgcgtccag ctcgttgagt ttctccagaa gcgttaatgt ctggcttctg
     4081 ataaagcggg ccatgttaag ggcggttttt tcctgtttgg tcactgatgc ctccgtgtaa
     4141 gggggatttc tgttcatggg ggtaatgata ccgatgaaac gagagaggat gctcacgata
     4201 cgggttactg atgatgaaca tgcccggtta ctggaacgtt gtgagggtaa acaactggcg
     4261 gtatggatgc ggcgggacca gagaaaaatc actcagggtc aatgccagcg cttcgttaat
     4321 acagatgtag gtgttccaca gggtagccag cagcatcctg cgatgcagat ccggaacata
     4381 atggtgcagg gcgctgactt ccgcgtttcc agactttacg aaacacggaa accgaagacc
     4441 attcatgttg ttgctcaggt cgcagacgtt ttgcagcagc agtcgcttca cgttcgctcg
     4501 cgtatcggtg attcattctg ctaaccagta aggcaacccc gccagcctag ccgggtcctc
     4561 aacgacagga gcacgatcat gcgcacccgt ggggccgcca tgccggcgat aatggcctgc
     4621 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     4681 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     4741 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     4801 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     4861 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     4921 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     4981 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     5041 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     5101 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     5161 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     5221 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     5281 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     5341 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     5401 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     5461 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     5521 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     5581 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     5641 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     5701 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     5761 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     5821 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     5881 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt ggctggcctg
     5941 gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga catcgtataa
     6001 cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct atcatgccat
     6061 accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct cccttatgcg
     6121 actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc gccgccgcaa
     6181 ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg cctgccacca
     6241 tacccacgc
//
