LOCUS       sequence-367538-        6084 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Encodes enzyme bovine carbonic anhydrase linked to Pep-H 
            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-Pep-H
FEATURES             Location/Qualifiers
     source          1..6084
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     966..985
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(1005..1082)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1288..1307
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1391..1410
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1391..1409
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1410..1434
                     /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             1449..1471
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2339..2356
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2409..2427)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2423..2470
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      2507..2962
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2594..2613)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2804..2825
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(3054..3869)
                     /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(3054..3869)
                     /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     3777..3796
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     3861..3880
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      3991..4579
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4733..4750
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4765..4904
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4830..4849)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4990..5012
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5006..5197)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6006..6084,1..1004))
                     /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 tgcgtattgg gcgccagggt ggtttttctt ttcaccagtg agacgggcaa cagctgattg
       61 cccttcaccg cctggccctg agagagttgc agcaagcggt ccacgctggt ttgccccagc
      121 aggcgaaaat cctgtttgat ggtggttaac ggcgggatat aacatgagct gtcttcggta
      181 tcgtcgtatc ccactaccga gatatccgca ccaacgcgca gcccggactc ggtaatggcg
      241 cgcattgcgc ccagcgccat ctgatcgttg gcaaccagca tcgcagtggg aacgatgccc
      301 tcattcagca tttgcatggt ttgttgaaaa ccggacatgg cactccagtc gccttcccgt
      361 tccgctatcg gctgaatttg attgcgagtg agatatttat gccagccagc cagacgcaga
      421 cgcgccgaga cagaacttaa tgggcccgct aacagcgcga tttgctggtg acccaatgcg
      481 accagatgct ccacgcccag tcgcgtaccg tcttcatggg agaaaataat actgttgatg
      541 ggtgtctggt cagagacatc aagaaataac gccggaacat tagtgcaggc agcttccaca
      601 gcaatggcat cctggtcatc cagcggatag ttaatgatca gcccactgac gcgttgcgcg
      661 agaagattgt gcaccgccgc tttacaggct tcgacgccgc ttcgttctac catcgacacc
      721 accacgctgg cacccagttg atcggcgcga gatttaatcg ccgcgacaat ttgcgacggc
      781 gcgtgcaggg ccagactgga ggtggcaacg ccaatcagca acgactgttt gcccgccagt
      841 tgttgtgcca cgcggttggg aatgtaattc agctccgcca tcgccgcttc cactttttcc
      901 cgcgttttcg cagaaacgtg gctggcctgg ttcaccacgc gggaaacggt ctgataagag
      961 acaccggcat actctgcgac atcgtataac gttactggtt tcacattcac caccctgaat
     1021 tgactctctt ccgggcgcta tcatgccata ccgcgaaagg ttttgcgcca ttcgatggtg
     1081 tccgggatct cgacgctctc ccttatgcga ctcctgcatt aggaagcagc ccagtagtag
     1141 gttgaggccg ttgagcaccg ccgccgcaag gaatggtgca tgcaaggaga tggcgcccaa
     1201 cagtcccccg gccacggggc ctgccaccat acccacgccg aaacaagcgc tcatgagccc
     1261 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
     1321 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
     1381 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
     1441 gaaataattt tgtttaactt taagaaggag atataccatg ggtatgtcac accattgggg
     1501 ctacggtaaa cacaacggtc cggaacactg gcacaaagac ttcccgattg ctaacggcga
     1561 acgtcagtca ccggtcgata ttgacaccaa agcagtggtt caggatccgg cactgaaacc
     1621 gctggctctg gtttatggcg aagctacgtc gcgtcgcatg gtcaacaatg gtcatagctt
     1681 taacgtggaa tatgatgact ctcaggataa agcagtgctg aaagacggcc cgctgaccgg
     1741 cacgtaccgt ctggttcagt ttcatttcca ctggggcagc tctgatgacc aaggttcaga
     1801 acacaccgtt gatcgcaaaa aatatgcggc cgaactgcat ctggtccact ggaacaccaa
     1861 atacggcgat tttggtaccg cagcgcagca accggatggt ctggccgttg tgggtgtttt
     1921 cctgaaagtc ggcgatgcga atccggccct gcagaaagtg ctggatgccc tggactcaat
     1981 caaaaccaaa ggcaaatcga cggattttcc gaacttcgac ccgggtagcc tgctgccgaa
     2041 tgttctggat tattggacct atccgggtag tctgaccacg ccgccgctgc tggaatccgt
     2101 gacgtggatt gttctgaaag aaccgatctc tgtgagttcc cagcaaatgc tgaaatttcg
     2161 taccctgaac ttcaatgccg aaggtgaacc ggaactgctg atgctggcca actggcgtcc
     2221 ggcacagccg ctgaaaaatc gtcaagtgcg cggcttcccg aaaggtggtg gtggtagtgg
     2281 cggcggtggc agttggttcc gttttgagtg ggagttcgag ttttggtaat aactcgagca
     2341 ccaccaccac caccactgag atccggctgc taacaaagcc cgaaaggaag ctgagttggc
     2401 tgctgccacc gctgagcaat aactagcata accccttggg gcctctaaac gggtcttgag
     2461 gggttttttg ctgaaaggag gaactatatc cggattggcg aatgggacgc gccctgtagc
     2521 ggcgcattaa gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc
     2581 gccctagcgc ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
     2641 ccccgtcaag ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
     2701 ctcgacccca aaaaacttga ttagggtgat ggttcacgta gtgggccatc gccctgatag
     2761 acggtttttc gccctttgac gttggagtcc acgttcttta atagtggact cttgttccaa
     2821 actggaacaa cactcaaccc tatctcggtc tattcttttg atttataagg gattttgccg
     2881 atttcggcct attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac
     2941 aaaatattaa cgcttacaat ttaggtggca cttttcgggg aaatgtgcgc ggaaccccta
     3001 tttgtttatt tttctaaata cattcaaata tgtatccgct catgaattaa ttcttagaaa
     3061 aactcatcga gcatcaaatg aaactgcaat ttattcatat caggattatc aataccatat
     3121 ttttgaaaaa gccgtttctg taatgaagga gaaaactcac cgaggcagtt ccataggatg
     3181 gcaagatcct ggtatcggtc tgcgattccg actcgtccaa catcaataca acctattaat
     3241 ttcccctcgt caaaaataag gttatcaagt gagaaatcac catgagtgac gactgaatcc
     3301 ggtgagaatg gcaaaagttt atgcatttct ttccagactt gttcaacagg ccagccatta
     3361 cgctcgtcat caaaatcact cgcatcaacc aaaccgttat tcattcgtga ttgcgcctga
     3421 gcgagacgaa atacgcgatc gctgttaaaa ggacaattac aaacaggaat cgaatgcaac
     3481 cggcgcagga acactgccag cgcatcaaca atattttcac ctgaatcagg atattcttct
     3541 aatacctgga atgctgtttt cccggggatc gcagtggtga gtaaccatgc atcatcagga
     3601 gtacggataa aatgcttgat ggtcggaaga ggcataaatt ccgtcagcca gtttagtctg
     3661 accatctcat ctgtaacatc attggcaacg ctacctttgc catgtttcag aaacaactct
     3721 ggcgcatcgg gcttcccata caatcgatag attgtcgcac ctgattgccc gacattatcg
     3781 cgagcccatt tatacccata taaatcagca tccatgttgg aatttaatcg cggcctagag
     3841 caagacgttt cccgttgaat atggctcata acaccccttg tattactgtt tatgtaagca
     3901 gacagtttta ttgttcatga ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc
     3961 agaccccgta gaaaagatca aaggatcttc ttgagatcct ttttttctgc gcgtaatctg
     4021 ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt tgtttgccgg atcaagagct
     4081 accaactctt tttccgaagg taactggctt cagcagagcg cagataccaa atactgtcct
     4141 tctagtgtag ccgtagttag gccaccactt caagaactct gtagcaccgc ctacatacct
     4201 cgctctgcta atcctgttac cagtggctgc tgccagtggc gataagtcgt gtcttaccgg
     4261 gttggactca agacgatagt taccggataa ggcgcagcgg tcgggctgaa cggggggttc
     4321 gtgcacacag cccagcttgg agcgaacgac ctacaccgaa ctgagatacc tacagcgtga
     4381 gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg
     4441 cagggtcgga acaggagagc gcacgaggga gcttccaggg ggaaacgcct rgtatcttta
     4501 tagtcctgtc gggtttcgcc acctctgact tgagcgtcga tttttgtgat gctcgtcagg
     4561 ggggcggagc ctatggaaaa acgccagcaa cgcggccttt ttacggttcc tggccttttg
     4621 ctggcctttt gctcacatgt tctttcctgc gttatcccct gattctgtgg ataaccgtat
     4681 taccgccttt gagtgagctg ataccgctcg ccgcagccga acgaccgagc gcagcgagtc
     4741 agtgagcgag gaagcggaag agcgcctgat gcggtatttt ctccttacgc atctgtgcgg
     4801 tatttcacac cgcaatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4861 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     4921 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4981 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     5041 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     5101 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     5161 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     5221 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     5281 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     5341 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     5401 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     5461 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     5521 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     5581 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     5641 ggagcacgat catgcgcacc cgtggggccg ccatgccggc gataatggcc tgcttctcgc
     5701 cgaaacgttt ggtggcggga ccagtgacga aggcttgagc gagggcgtgc aagattccga
     5761 ataccgcaag cgacaggccg atcatcgtcg cgctccagcg aaagcggtcc tcgccgaaaa
     5821 tgacccagag cgctgccggc acctgtccta cgagttgcat gataaagaag acagtcataa
     5881 gtgcggcgac gatagtcatg ccccgcgccc accggaagga gctgactggg ttgaaggctc
     5941 tcaagggcat cggtcgagat cccggtgcct aatgagtgag ctaacttaca ttaattgcgt
     6001 tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg
     6061 gccaacgcgc ggggagaggc ggtt
//
