LOCUS       sequence-217773-        5681 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  protein purification.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5681)
  TITLE     plasmids for expression of yeast spliceosomal proteins
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5681)
  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            "Unpublished"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: pET30a-Spp2
FEATURES             Location/Qualifiers
     source          1..5681
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     50..69
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        50..68
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    69..93
                     /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             108..130
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             591..608
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(661..679)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      675..722
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      759..1214
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(846..865)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     1056..1077
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(1306..2121)
                     /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(1306..2121)
                     /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     2029..2048
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     2113..2132
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      2243..2831
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2732..2751
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2985..3002
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3017..3156
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3082..3101)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3242..3264
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3258..3449)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(4258..5340)
                     /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     5302..5321
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(5341..5418)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     5624..5643
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 acgatgcgtc cggcgtagag gatcgagatc gatctcgatc ccgcgaaatt aatacgactc
       61 actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt gtttaacttt
      121 aagaaggaga tatacatatg gcggaaactg caagcctttc gcataaacct caaagtaaga
      181 taaagattca aagtattgat aaattcgatc ttgatgaaga atcatcttct aagaagaaat
      241 tagtgatcaa gctgagtgaa aatgcggata ccaaaaaaaa cgatgctcct ctggtggaat
      301 atgtcaccga aaaagaatat aatgaggtac ctgtcgagga atttggagat gctttacttc
      361 ggggtatggg gtgggaaagt gactccgagc aagattctaa aggcgacaag acacagagtc
      421 gaaacaagga cgtttcaaat gtttcacaga tacatcctga tggactggga attggtgcca
      481 agttaaataa ggcaataaac gtggaggagg cttcatttat gcccgtcgta aaaatcgata
      541 aaataacggg cactaaagtt gatgacgaca aaaaaaataa gtcactcgag caccaccacc
      601 accaccactg agatccggct gctaacaaag cccgaaagga agctgagttg gctgctgcca
      661 ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg aggggttttt
      721 tgctgaaagg aggaactata tccggattgg cgaatgggac gcgccctgta gcggcgcatt
      781 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
      841 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
      901 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
      961 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     1021 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     1081 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     1141 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaatttta acaaaatatt
     1201 aacgcttaca atttaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     1261 tttttctaaa tacattcaaa tatgtatccg ctcatgaatt aattcttaga aaaactcatc
     1321 gagcatcaaa tgaaactgca atttattcat atcaggatta tcaataccat atttttgaaa
     1381 aagccgtttc tgtaatgaag gagaaaactc accgaggcag ttccatagga tggcaagatc
     1441 ctggtatcgg tctgcgattc cgactcgtcc aacatcaata caacctatta atttcccctc
     1501 gtcaaaaata aggttatcaa gtgagaaatc accatgagtg acgactgaat ccggtgagaa
     1561 tggcaaaagt ttatgcattt ctttccagac ttgttcaaca ggccagccat tacgctcgtc
     1621 atcaaaatca ctcgcatcaa ccaaaccgtt attcattcgt gattgcgcct gagcgagacg
     1681 aaatacgcga tcgctgttaa aaggacaatt acaaacagga atcgaatgca accggcgcag
     1741 gaacactgcc agcgcatcaa caatattttc acctgaatca ggatattctt ctaatacctg
     1801 gaatgctgtt ttcccgggga tcgcagtggt gagtaaccat gcatcatcag gagtacggat
     1861 aaaatgcttg atggtcggaa gaggcataaa ttccgtcagc cagtttagtc tgaccatctc
     1921 atctgtaaca tcattggcaa cgctaccttt gccatgtttc agaaacaact ctggcgcatc
     1981 gggcttccca tacaatcgat agattgtcgc acctgattgc ccgacattat cgcgagccca
     2041 tttataccca tataaatcag catccatgtt ggaatttaat cgcggcctag agcaagacgt
     2101 ttcccgttga atatggctca taacacccct tgtattactg tttatgtaag cagacagttt
     2161 tattgttcat gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg
     2221 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     2281 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     2341 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc cttctagtgt
     2401 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     2461 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     2521 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     2581 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     2641 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     2701 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     2761 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     2821 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     2881 ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct
     2941 ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg
     3001 aggaagcgga agagcgcctg atgcggtatt ttctccttac gcatctgtgc ggtatttcac
     3061 accgcaatgg tgcactctca gtacaatctg ctctgatgcc gcatagttaa gccagtatac
     3121 actccgctat cgctacgtga ctgggtcatg gctgcgcccc gacacccgcc aacacccgct
     3181 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccgtc
     3241 tccgggagct gcatgtgtca gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg
     3301 cggtaaagct catcagcgtg gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg
     3361 tccagctcgt tgagtttctc cagaagcgtt aatgtctggc ttctgataaa gcgggccatg
     3421 ttaagggcgg ttttttcctg tttggtcact gatgcctccg tgtaaggggg atttctgttc
     3481 atgggggtaa tgataccgat gaaacgagag aggatgctca cgatacgggt tactgatgat
     3541 gaacatgccc ggttactgga acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg
     3601 gaccagagaa aaatcactca gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt
     3661 ccacagggta gccagcagca tcctgcgatg cagatccgga acataatggt gcagggcgct
     3721 gacttccgcg tttccagact ttacgaaaca cggaaaccga agaccattca tgttgttgct
     3781 caggtcgcag acgttttgca gcagcagtcg cttcacgttc gctcgcgtat cggtgattca
     3841 ttctgctaac cagtaaggca accccgccag cctagccggg tcctcaacga caggagcacg
     3901 atcatgcgca cccgtggggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     3961 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     4021 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     4081 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     4141 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     4201 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     4261 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     4321 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     4381 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     4441 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     4501 tgagctgtct tcggtatcgt cgtatcccac taccgagatg tccgcaccaa cgcgcagccc
     4561 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     4621 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     4681 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcca
     4741 gccagccaga cgcagacgcg ccgagacaga acttaatggg cccgctaaca gcgcgatttg
     4801 ctggtgaccc aatgcgacca gatgctccac gcccagtcgc gtaccgtctt catgggagaa
     4861 aataatactg ttgatgggtg tctggtcaga gacatcaaga aataacgccg gaacattagt
     4921 gcaggcagct tccacagcaa tggcatcctg gtcatccagc ggatagttaa tgatcagccc
     4981 actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta caggcttcga cgccgcttcg
     5041 ttctaccatc gacaccacca cgctggcacc cagttgatcg gcgcgagatt taatcgccgc
     5101 gacaatttgc gacggcgcgt gcagggccag actggaggtg gcaacgccaa tcagcaacga
     5161 ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg taattcagct ccgccatcgc
     5221 cgcttccact ttttcccgcg ttttcgcaga aacgtggctg gcctggttca ccacgcggga
     5281 aacggtctga taagagacac cggcatactc tgcgacatcg tataacgtta ctggtttcac
     5341 attcaccacc ctgaattgac tctcttccgg gcgctatcat gccataccgc gaaaggtttt
     5401 gcgccattcg atggtgtccg ggatctcgac gctctccctt atgcgactcc tgcattagga
     5461 agcagcccag tagtaggttg aggccgttga gcaccgccgc cgcaaggaat ggtgcatgca
     5521 aggagatggc gcccaacagt cccccggcca cggggcctgc caccataccc acgccgaaac
     5581 aagcgctcat gagcccgaag tggcgagccc gatcttcccc atcggtgatg tcggcgatat
     5641 aggcgccagc aaccgcacct gtggcgccgg tgatgccggc c
//
