LOCUS       sequence-257694-        5622 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of ranaspumin 2 as a His-tagged fusion protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5622)
  AUTHORS   Mackenzie CD, Smith BO, Meister A, Blume A, Zhao X, Lu JR, Kennedy 
            MW, Cooper A
  TITLE     Ranaspumin-2: structure and function of a surfactant protein from 
            the foam nests of a tropical frog.
  JOURNAL   Biophys J. 2009 Jun 17;96(12):4984-92. doi: 
            10.1016/j.bpj.2009.03.044.
  PUBMED    19527658
REFERENCE   2  (bases 1 to 5622)
  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"; doi: 
            "10.1016/j.bpj.2009.03"; journalName: "Biophys J"; date: 
            "2009-06-17- 17"; volume: "96"; issue: "12"; pages: "4984-92"
COMMENT     Sequence Label: pET28-Rsn2
FEATURES             Location/Qualifiers
     source          1..5622
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..20
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1..19
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    20..44
                     /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             59..81
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             100..117
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             127..144
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             487..504
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(557..575)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      571..618
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      655..1110
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(742..761)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     952..973
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1202..2017)
                     /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(1202..2017)
                     /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     1925..1944
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2009..2028
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2139..2727
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2628..2647
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2881..2898
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2913..3052
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2978..2997)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3138..3160
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3154..3345)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4154..5236)
                     /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     5198..5217
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5237..5314)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5520..5539
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta gaaataattt
       61 tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca tcatcacagc
      121 agcggcctgg tgccgcgcgg cagccatatg ttaatattag atggggacct actaaaggac
      181 aagttaaagt taccggtcat tgacaatctg tttggtaaag aactccttga caagtttcaa
      241 gatgatatta aggacaaata tggggtggac accaaagacc ttaagatcct caagacatca
      301 gaagataaaa ggttttacta tgtgtcggtc gatgctggag atggcgagaa atgtaaattc
      361 aagattagaa aagatgtcga tgttccaaag atggtcggac gcaaatgtag gaaggacgat
      421 gatgatgatg atggatatta ggatccgaat tcgagctccg tcgacaagct tgcggccgca
      481 ctcgagcacc accaccacca ccactgagat ccggctgcta acaaagcccg aaaggaagct
      541 gagttggctg ctgccaccgc tgagcaataa ctagcataac cccttggggc ctctaaacgg
      601 gtcttgaggg gttttttgct gaaaggagga actatatccg gattggcgaa tgggacgcgc
      661 cctgtagcgg cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac
      721 ttgccagcgc cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg
      781 ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt
      841 tacggcacct cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc
      901 cctgatagac ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct
      961 tgttccaaac tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga
     1021 ttttgccgat ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga
     1081 attttaacaa aatattaacg cttacaattt aggtggcact tttcggggaa atgtgcgcgg
     1141 aacccctatt tgtttatttt tctaaataca ttcaaatatg tatccgctca tgaattaatt
     1201 cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa
     1261 taccatattt ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc
     1321 ataggatggc aagatcctgg tatcggtctg cgattccgac tcgtccaaca tcaatacaac
     1381 ctattaattt cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga
     1441 ctgaatccgg tgagaatggc aaaagtttat gcatttcttt ccagacttgt tcaacaggcc
     1501 agccattacg ctcgtcatca aaatcactcg catcaaccaa accgttattc attcgtgatt
     1561 gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg acaattacaa acaggaatcg
     1621 aatgcaaccg gcgcaggaac actgccagcg catcaacaat attttcacct gaatcaggat
     1681 attcttctaa tacctggaat gctgttttcc cggggatcgc agtggtgagt aaccatgcat
     1741 catcaggagt acggataaaa tgcttgatgg tcggaagagg cataaattcc gtcagccagt
     1801 ttagtctgac catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa
     1861 acaactctgg cgcatcgggc ttcccataca atcgatagat tgtcgcacct gattgcccga
     1921 cattatcgcg agcccattta tacccatata aatcagcatc catgttggaa tttaatcgcg
     1981 gcctagagca agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta
     2041 tgtaagcaga cagttttatt gttcatgacc aaaatccctt aacgtgagtt ttcgttccac
     2101 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     2161 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     2221 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     2281 actgtccttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     2341 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     2401 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     2461 gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     2521 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     2581 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     2641 tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc
     2701 tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg
     2761 gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat
     2821 aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc
     2881 agcgagtcag tgagcgagga agcggaagag cgcctgatgc ggtattttct ccttacgcat
     2941 ctgtgcggta tttcacaccg caatggtgca ctctcagtac aatctgctct gatgccgcat
     3001 agttaagcca gtatacactc cgctatcgct acgtgactgg gtcatggctg cgccccgaca
     3061 cccgccaaca cccgctgacg cgccctgacg ggcttgtctg ctcccggcat ccgcttacag
     3121 acaagctgtg accgtctccg ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa
     3181 acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg tgaagcgatt cacagatgtc
     3241 tgcctgttca tccgcgtcca gctcgttgag tttctccaga agcgttaatg tctggcttct
     3301 gataaagcgg gccatgttaa gggcggtttt ttcctgtttg gtcactgatg cctccgtgta
     3361 agggggattt ctgttcatgg gggtaatgat accgatgaaa cgagagagga tgctcacgat
     3421 acgggttact gatgatgaac atgcccggtt actggaacgt tgtgagggta aacaactggc
     3481 ggtatggatg cggcgggacc agagaaaaat cactcagggt caatgccagc gcttcgttaa
     3541 tacagatgta ggtgttccac agggtagcca gcagcatcct gcgatgcaga tccggaacat
     3601 aatggtgcag ggcgctgact tccgcgtttc cagactttac gaaacacgga aaccgaagac
     3661 cattcatgtt gttgctcagg tcgcagacgt tttgcagcag cagtcgcttc acgttcgctc
     3721 gcgtatcggt gattcattct gctaaccagt aaggcaaccc cgccagccta gccgggtcct
     3781 caacgacagg agcacgatca tgcgcacccg tggggccgcc atgccggcga taatggcctg
     3841 cttctcgccg aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa
     3901 gattccgaat accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc
     3961 gccgaaaatg acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac
     4021 agtcataagt gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt
     4081 gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt
     4141 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     4201 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc
     4261 ttttcaccag tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt
     4321 gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta
     4381 acggcgggat ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg
     4441 caccaacgcg cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt
     4501 tggcaaccag catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa
     4561 aaccggacat ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag
     4621 tgagatattt atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg
     4681 ctaacagcgc gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac
     4741 cgtcttcatg ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata
     4801 acgccggaac attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat
     4861 agttaatgat cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg
     4921 cttcgacgcc gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc
     4981 gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa
     5041 cgccaatcag caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat
     5101 tcagctccgc catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct
     5161 ggttcaccac gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata
     5221 acgttactgg tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca
     5281 taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc
     5341 gactcctgca ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca
     5401 aggaatggtg catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc
     5461 atacccacgc cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg
     5521 gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg
     5581 atgcgtccgg cgtagaggat cgagatctcg atcccgcgaa at
//
