LOCUS       sequence-259998-        5967 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  bacterial expression of ranaspumin 2 as a fusion protein with the 
            fluorescent tag iLOV.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5967)
  TITLE     Brian Smith Plasmids
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5967)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pET28-iLOV-Rsn2
FEATURES             Location/Qualifiers
     source          1..5967
                     /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             832..849
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(902..920)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      916..963
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      1000..1455
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(1087..1106)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1297..1318
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1547..2362)
                     /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(1547..2362)
                     /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     2270..2289
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2354..2373
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2484..3072
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2973..2992
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3226..3243
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3258..3397
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3323..3342)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3483..3505
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3499..3690)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4499..5581)
                     /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     5543..5562
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5582..5659)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5865..5884
                     /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 atcggtacca ttgagaagaa tttcgtaatc
      181 actgatccac gtctgccgga caacccgatc atcttcgctt ccgacggctt cctggagctg
      241 accgagtatt cccgtgaaga gatcctgggc cgtaacgccc gtttcctgca gggcccggag
      301 actgatcagg cgacggttca gaaaattcgc gatgctatcc gtgaccagcg tgaaactacg
      361 gtgcaactga ttaactacac gaaaagcggt aagaaattct ggaacctgct gcatctgcaa
      421 ccagtccgcg atcagaaagg cgagctgcag tatttcatcg gcgttcaact ggatggttct
      481 gaccacgtac atatgttaat attagatggg gacctactaa aggacaagtt aaagttaccg
      541 gtcattgaca atctgtttgg taaagaactc cttgacaagt ttcaagatga tattaaggac
      601 aaatatgggg tggacaccaa agaccttaag atcctcaaga catcagaaga taaaaggttt
      661 tactatgtgt cggtcgatgc tggagatggc gagaaatgta aattcaagat tagaaaagat
      721 gtcgatgttc caaagatggt cggacgcaaa tgtaggaagg acgatgatga tgatgatgga
      781 tattaggatc cgaattcgag ctccgtcgac aagcttgcgg ccgcactcga gcaccaccac
      841 caccaccact gagatccggc tgctaacaaa gcccgaaagg aagctgagtt ggctgctgcc
      901 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
      961 ttgctgaaag gaggaactat atccggattg gcgaatggga cgcgccctgt agcggcgcat
     1021 taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc tacacttgcc agcgccctag
     1081 cgcccgctcc tttcgctttc ttcccttcct ttctcgccac gttcgccggc tttccccgtc
     1141 aagctctaaa tcgggggctc cctttagggt tccgatttag tgctttacgg cacctcgacc
     1201 ccaaaaaact tgattagggt gatggttcac gtagtgggcc atcgccctga tagacggttt
     1261 ttcgcccttt gacgttggag tccacgttct ttaatagtgg actcttgttc caaactggaa
     1321 caacactcaa ccctatctcg gtctattctt ttgatttata agggattttg ccgatttcgg
     1381 cctattggtt aaaaaatgag ctgatttaac aaaaatttaa cgcgaatttt aacaaaatat
     1441 taacgcttac aatttaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt
     1501 atttttctaa atacattcaa atatgtatcc gctcatgaat taattcttag aaaaactcat
     1561 cgagcatcaa atgaaactgc aatttattca tatcaggatt atcaatacca tatttttgaa
     1621 aaagccgttt ctgtaatgaa ggagaaaact caccgaggca gttccatagg atggcaagat
     1681 cctggtatcg gtctgcgatt ccgactcgtc caacatcaat acaacctatt aatttcccct
     1741 cgtcaaaaat aaggttatca agtgagaaat caccatgagt gacgactgaa tccggtgaga
     1801 atggcaaaag tttatgcatt tctttccaga cttgttcaac aggccagcca ttacgctcgt
     1861 catcaaaatc actcgcatca accaaaccgt tattcattcg tgattgcgcc tgagcgagac
     1921 gaaatacgcg atcgctgtta aaaggacaat tacaaacagg aatcgaatgc aaccggcgca
     1981 ggaacactgc cagcgcatca acaatatttt cacctgaatc aggatattct tctaatacct
     2041 ggaatgctgt tttcccgggg atcgcagtgg tgagtaacca tgcatcatca ggagtacgga
     2101 taaaatgctt gatggtcgga agaggcataa attccgtcag ccagtttagt ctgaccatct
     2161 catctgtaac atcattggca acgctacctt tgccatgttt cagaaacaac tctggcgcat
     2221 cgggcttccc atacaatcga tagattgtcg cacctgattg cccgacatta tcgcgagccc
     2281 atttataccc atataaatca gcatccatgt tggaatttaa tcgcggccta gagcaagacg
     2341 tttcccgttg aatatggctc ataacacccc ttgtattact gtttatgtaa gcagacagtt
     2401 ttattgttca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     2461 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     2521 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     2581 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     2641 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     2701 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     2761 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     2821 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     2881 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     2941 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     3001 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     3061 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     3121 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     3181 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     3241 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     3301 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     3361 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     3421 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     3481 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     3541 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     3601 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     3661 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     3721 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     3781 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     3841 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     3901 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     3961 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     4021 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     4081 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     4141 gatcatgcgc acccgtgggg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     4201 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     4261 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     4321 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     4381 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     4441 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     4501 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     4561 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     4621 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     4681 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     4741 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     4801 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     4861 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     4921 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     4981 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     5041 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     5101 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     5161 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     5221 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     5281 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     5341 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     5401 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     5461 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     5521 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     5581 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     5641 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     5701 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     5761 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     5821 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
     5881 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
     5941 aggatcgaga tctcgatccc gcgaaat
//
