LOCUS       sequence-426091-        6010 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses SUMO-tagged RvSAHS4 protein from tardigrade R. 
            varieornatus in E. coli.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6010)
  TITLE     Bacterial expression plasmids for production of recombinant 
            SUMO-tagged tardigrade SAHS proteins
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6010)
  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            "Unpublished"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: PAS_RvSAHS4
FEATURES             Location/Qualifiers
     source          1..6010
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(517..535)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      531..578
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      615..1070
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(702..721)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     912..933
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1162..1977)
                     /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(1162..1977)
                     /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     1885..1904
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     1969..1988
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2099..2687
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2588..2607
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2841..2858
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    2873..3012
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(2938..2957)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3098..3120
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3114..3305)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4114..5196)
                     /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     5158..5177
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5197..5274)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5480..5499
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     5587..5606
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        5587..5605
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    5610..5626
                     /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             5645..5667
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             5681..5710
                     /codon_start=1
                     /product="10xHis affinity tag"
                     /label=10xHis
                     /translation="HHHHHHHHHH"
ORIGIN
        1 cgccctcatg acgagagcaa ggcccagtgg accggcaaac cttggcttgg taagtgggag
       61 tcgattgatg gcacgcctga aaactgggag gcatttgtga aggcggcgaa catcccccca
      121 aaggaccaag ccctgtacaa cgggaagcag aagaccttgt tgaagtattg gaaagaggcg
      181 ggtgaagacc attatcacgt gcaaacctcc ttccctggga cggaacataa gatggaaacc
      241 agttttaaga tgggccagga aggaactctg tcgcatgatg gcgtcgattt aaaatatgtt
      301 tgtaccgaag atggtgagca gttaataacc aaaatcaaca ttccatctaa gaatcaggaa
      361 acaattgtca cttacacagc tacgggtgat gatctcgagc agacgtttac cagcaatggt
      421 gttacgggaa agcgttggta caaaaagatc catgcctaat gatcgaggat ccggctgcta
      481 acaaagcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa ctagcataac
      541 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
      601 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
      661 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
      721 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
      781 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
      841 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
      901 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
      961 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     1021 ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt aggtggcact
     1081 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     1141 tatccgctca tgaattaatt cttagaaaaa ctcatcgagc atcaaatgaa actgcaattt
     1201 attcatatca ggattatcaa taccatattt ttgaaaaagc cgtttctgta atgaaggaga
     1261 aaactcaccg aggcagttcc ataggatggc aagatcctgg tatcggtctg cgattccgac
     1321 tcgtccaaca tcaatacaac ctattaattt cccctcgtca aaaataaggt tatcaagtga
     1381 gaaatcacca tgagtgacga ctgaatccgg tgagaatggc aaaagtttat gcatttcttt
     1441 ccagacttgt tcaacaggcc agccattacg ctcgtcatca aaatcactcg catcaaccaa
     1501 accgttattc attcgtgatt gcgcctgagc gagacgaaat acgcgatcgc tgttaaaagg
     1561 acaattacaa acaggaatcg aatgcaaccg gcgcaggaac actgccagcg catcaacaat
     1621 attttcacct gaatcaggat attcttctaa tacctggaat gctgttttcc cggggatcgc
     1681 agtggtgagt aaccatgcat catcaggagt acggataaaa tgcttgatgg tcggaagagg
     1741 cataaattcc gtcagccagt ttagtctgac catctcatct gtaacatcat tggcaacgct
     1801 acctttgcca tgtttcagaa acaactctgg cgcatcgggc ttcccataca atcgatagat
     1861 tgtcgcacct gattgcccga cattatcgcg agcccattta tacccatata aatcagcatc
     1921 catgttggaa tttaatcgcg gcctagagca agacgtttcc cgttgaatat ggctcataac
     1981 accccttgta ttactgttta tgtaagcaga cagttttatt gttcatgacc aaaatccctt
     2041 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2101 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2161 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2221 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     2281 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     2341 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     2401 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     2461 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     2521 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     2581 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     2641 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     2701 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     2761 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     2821 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     2881 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     2941 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     3001 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3061 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     3121 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     3181 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     3241 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     3301 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     3361 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     3421 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     3481 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     3541 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     3601 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     3661 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     3721 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggggccgcc
     3781 atgccggcga taatggcctg cttctcgccg aaacgtttgg tggcgggacc agtgacgaag
     3841 gcttgagcga gggcgtgcaa gattccgaat accgcaagcg acaggccgat catcgtcgcg
     3901 ctccagcgaa agcggtcctc gccgaaaatg acccagagcg ctgccggcac ctgtcctacg
     3961 agttgcatga taaagaagac agtcataagt gcggcgacga tagtcatgcc ccgcgcccac
     4021 cggaaggagc tgactgggtt gaaggctctc aagggcatcg gtcgagatcc cggtgcctaa
     4081 tgagtgagct aacttacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac
     4141 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt
     4201 gggcgccagg gtggtttttc ttttcaccag tgagacgggc aacagctgat tgcccttcac
     4261 cgcctggccc tgagagagtt gcagcaagcg gtccacgctg gtttgcccca gcaggcgaaa
     4321 atcctgtttg atggtggtta acggcgggat ataacatgag ctgtcttcgg tatcgtcgta
     4381 tcccactacc gagatgtccg caccaacgcg cagcccggac tcggtaatgg cgcgcattgc
     4441 gcccagcgcc atctgatcgt tggcaaccag catcgcagtg ggaacgatgc cctcattcag
     4501 catttgcatg gtttgttgaa aaccggacat ggcactccag tcgccttccc gttccgctat
     4561 cggctgaatt tgattgcgag tgagatattt atgccagcca gccagacgca gacgcgccga
     4621 gacagaactt aatgggcccg ctaacagcgc gatttgctgg tgacccaatg cgaccagatg
     4681 ctccacgccc agtcgcgtac cgtcttcatg ggagaaaata atactgttga tgggtgtctg
     4741 gtcagagaca tcaagaaata acgccggaac attagtgcag gcagcttcca cagcaatggc
     4801 atcctggtca tccagcggat agttaatgat cagcccactg acgcgttgcg cgagaagatt
     4861 gtgcaccgcc gctttacagg cttcgacgcc gcttcgttct accatcgaca ccaccacgct
     4921 ggcacccagt tgatcggcgc gagatttaat cgccgcgaca atttgcgacg gcgcgtgcag
     4981 ggccagactg gaggtggcaa cgccaatcag caacgactgt ttgcccgcca gttgttgtgc
     5041 cacgcggttg ggaatgtaat tcagctccgc catcgccgct tccacttttt cccgcgtttt
     5101 cgcagaaacg tggctggcct ggttcaccac gcgggaaacg gtctgataag agacaccggc
     5161 atactctgcg acatcgtata acgttactgg tttcacattc accaccctga attgactctc
     5221 ttccgggcgc tatcatgcca taccgcgaaa ggttttgcgc cattcgatgg tgtccgggat
     5281 ctcgacgctc tcccttatgc gactcctgca ttaggaagca gcccagtagt aggttgaggc
     5341 cgttgagcac cgccgccgca aggaatggtg catgcaagga gatggcgccc aacagtcccc
     5401 cggccacggg gcctgccacc atacccacgc cgaaacaagc gctcatgagc ccgaagtggc
     5461 gagcccgatc ttccccatcg gtgatgtcgg cgatataggc gccagcaacc gcacctgtgg
     5521 cgccggtgat gccggccacg atgcgtccgg cgtagaggat cgagatcgat ctcgatcccg
     5581 cgaaattaat acgactcact atagggaaat tgtgagcgga taacaattcc cctctagaaa
     5641 taattttgtt taactttaag aaggagatat acatatgggt caccaccacc accaccacca
     5701 ccaccaccac ggtagcctgc aagattcgga agtcaaccaa gaagcaaagc cggaagtcaa
     5761 gccggaagtg aaaccggaaa cccatattaa cctgaaagtt agtgacggca gctctgaaat
     5821 tttctttaag atcaaaaaga ccacgccgct gcgtcgcctg atggaagcgt ttgccaaacg
     5881 tcagggcaag gaaatggata gcctgcgttt cctgtatgac ggtattcgca tccaggcaga
     5941 tcaagcgccg gaagacctgg acatggaaga caacgacatc attgaagccc accgtgaaca
     6001 gattggcggc
//
