LOCUS       sequence-345508-        8041 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of His14-MBP-bdSUMO-CRISPR associated 
            IscB-TwinStrep.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8041)
  AUTHORS   Altae-Tran H, Kannan S, Demircioglu FE, Oshiro R, Nety SP, McKay LJ,
            Dlakic M, Inskeep WP, Makarova KS, Macrae RK, Koonin EV, Zhang F
  TITLE     The widespread IS200/IS605 transposon family encodes diverse 
            programmable RNA-guided endonucleases.
  JOURNAL   Science. 2021 Oct;374(6563):57-65. doi: 10.1126/science.abj6856. 
            Epub 2021 Sep 9.
  PUBMED    34591643
REFERENCE   2  (bases 1 to 8041)
  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: 
            "10.1126/science.abj6856"; journalName: "Science"; date: "2021-10"; 
            volume: "374"; issue: "6563"; pages: "57-65"
COMMENT     Sequence Label: pHS0570 pET45b(+) T7/LacO His14-MBP-bdSUMO-CRI...
FEATURES             Location/Qualifiers
     source          1..8041
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1067..1258
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(1252..1274)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    1360..1499
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     1415..1434
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(1514..1531)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1685..2273)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(1765..1784)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        2354..2446
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2447..2515,2516..3307)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(2665..2684)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      complement(3422..3877)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3559..3580)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3771..3790
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     terminator      complement(3914..3961)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     3957..3975
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(join(4001..4024,4025..4060,4061..4084))
                     /codon_start=1
                     /product="two Strep-Tag(R)II moieties connected by a linker
                     for enhanced binding to Strep-Tactin(R), an engineered form
                     of streptavidin (Schmidt et al., 2013)"
                     /label=Twin-Strep-tag
                     /translation="WSHPQFEKGGGSGGGSGGSAWSHPQFEK"
     CDS             complement(4097..4114)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(5801..6898)
                     /codon_start=1
                     /gene="malE (mutated)"
                     /product="maltose binding protein from E. coli"
                     /label=MBP
                     /note="This version of the gene does not encode a signal 
                     sequence, so MBP will remain in the cytosol."
                     /translation="KTEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEE
                     KFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLI
                     AYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAA
                     DGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGET
                     AMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLE
                     NYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFW
                     YAVRTAVINAASGRQTVDEALKDAQT"
     primer_bind     complement(5804..5827)
                     /label=MBP-F
                     /note="Maltose binding protein, forward primer"
     regulatory      5935..5944
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     RBS             6983..7005
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    7020..7044
                     /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)."
     primer_bind     complement(7044..7063)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(7045..7063)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(7151..7170)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        7376..7453
                     /gene="lacI"
                     /label=lacI promoter
     CDS             join(7454..8041,1..495)
                     /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     complement(7473..7492)
                     /label=LacI-R
                     /note="LacI, reverse primer"
ORIGIN
        1 cgtctggctg gctggcataa atatctcact cgcaatcaaa ttcagccgat agcggaacgg
       61 gaaggcgact ggagtgccat gtccggtttt caacaaacca tgcaaatgct gaatgagggc
      121 atcgttccca ctgcgatgct ggttgccaac gatcagatgg cgctgggcgc aatgcgcgcc
      181 attaccgagt ccgggctgcg cgttggtgcg gacatctcgg tagtgggata cgacgatacc
      241 gaagacagct catgttatat cccgccgtta accaccatca aacaggattt tcgcctgctg
      301 gggcaaacca gcgtggaccg cttgctgcaa ctctctcagg gccaggcggt gaagggcaat
      361 cagctgttgc ccgtctcact ggtgaaaaga aaaaccaccc tggcgcccaa tacgcaaacc
      421 gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg
      481 gaaagcgggc agtgagcgca acgcaattaa tgtaagttag ctcactcatt aggcaccggg
      541 atctcgaccg atgcccttga gagccttcaa cccagtcagc tccttccggt gggcgcgggg
      601 catgactagc atgatcgtgc tcctgtcgtt gaggacccgg ctaggctggc ggggttgcct
      661 tactggttag cagaatgaat caccgatacg cgagcgaacg tgaagcgact gctgctgcaa
      721 aacgtctgcg acctgagcaa caacatgaat ggtcttcggt ttccgtgttt cgtaaagtct
      781 ggaaacgcgg aagtcagcgc cctgcaccat tatgttccgg atctgcatcg caggatgctg
      841 ctggctaccc tgtggaacac ctacatctgt attaacgaag cgctggcatt gaccctgagt
      901 gatttttctc tggtcccgcc gcatccatac cgccagttgt ttaccctcac aacgttccag
      961 taaccgggca tgttcatcat cagtaacccg tatcgtgagc atcctctctc gtttcatcgg
     1021 tatcattacc cccatgaaca gaaatccccc ttacacggag gcatcagtga ccaaacagga
     1081 aaaaaccgcc cttaacatgg cccgctttat cagaagccag acattaacgc ttctggagaa
     1141 actcaacgag ctggacgcgg atgaacaggc agacatctgt gaatcgcttc acgaccacgc
     1201 tgatgagctt taccgcagct gcctcgcgcg tttcggtgat gacggtgaaa acctctgaca
     1261 catgcagctc ccggagacgg tcacagcttg tctgtaagcg gatgccggga gcagacaagc
     1321 ccgtcagggc gcgtcagcgg gtgttggcgg gtgtcggggc gcagccatga cccagtcacg
     1381 tagcgatagc ggagtgtata ctggcttaac tatgcggcat cagagcagat tgtactgaga
     1441 gtgcaccatt gcggtgtgaa ataccgcaca gatgcgtaag gagaaaatac cgcatcaggc
     1501 gctcttccgc ttcctcgctc actgactcgc tgcgctcggt cgttcggctg cggcgagcgg
     1561 tatcagctca ctcaaaggcg gtaatacggt tatccacaga atcaggggat aacgcaggaa
     1621 agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg
     1681 cgtttttcca taggctccgc ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga
     1741 ggtggcgaaa cccgacagga ctataaagat accaggcgtt tccccctgga agctccctcg
     1801 tgcgctctcc tgttccgacc ctgccgctta ccggatacct gtccgccttt ctcccttcgg
     1861 gaagcgtggc gctttctcat agctcacgct gtaggtatct cagttcggtg taggtcgttc
     1921 gctccaagct gggctgtgtg cacgaacccc ccgttcagcc cgaccgctgc gccttatccg
     1981 gtaactatcg tcttgagtcc aacccggtaa gacacgactt atcgccactg gcagcagcca
     2041 ctggtaacag gattagcaga gcgaggtatg taggcggtgc tacagagttc ttgaagtggt
     2101 ggcctaacta cggctacact agaaggacag tatttggtat ctgcgctctg ctgaagccag
     2161 ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa acaaaccacc gctggtagcg
     2221 gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa aaaaggatct caagaagatc
     2281 ctttgatctt ttctacgggg tctgacgctc agtggaacga aaactcacgt taagggattt
     2341 tggtcatgac ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     2401 caataaccct gataaatgct tcaatcatga ttgaaaaagg aagagtatga gtattcaaca
     2461 tttccgtgtc gcccttattc ccttttttgc ggcattttgc cttcctgttt ttgctcaccc
     2521 agaaacgctg gtgaaagtaa aagatgctga agatcagttg ggtgcacgag tgggttacat
     2581 cgaactggat ctcaacagcg gtaagatcct tgagagtttt cgccccgaag aacgttttcc
     2641 aatgatgagc acttttaaag ttctgctatg tggcgcggta ttatcccgta ttgacgccgg
     2701 gcaagagcaa ctcggtcgcc gcatacacta ttctcagaat gacttggttg agtactcacc
     2761 agtcacagaa aagcatctta cggatggcat gacagtaaga gaattatgca gtgctgccat
     2821 aaccatgagt gataacactg cggccaactt acttctgaca acgatcggag gaccgaagga
     2881 gctaaccgct tttttgcaca acatggggga tcatgtaact cgccttgatc gttgggaacc
     2941 ggagctgaat gaagccatac caaacgacga gcgtgacacc acgatgcctg tagcaatggc
     3001 aacaacgttg cgcaaactat taactggcga actacttact ctagcttccc ggcaacaatt
     3061 aatagactgg atggaggcgg ataaagttgc aggaccactt ctgcgctcgg cccttccggc
     3121 tggctggttt attgctgata aatctggagc cggtgagcgt gggtctcgcg gtatcattgc
     3181 agcactgggg ccagatggta agccctcccg tatcgtagtt atctacacga cggggagtca
     3241 ggcaactatg gatgaacgaa atagacagat cgctgagata ggtgcctcac tgattaagca
     3301 ttggtaactg tcagaccaag tttactcata tatactttag attgatttaa aacttcattt
     3361 ttaatttaaa aggatctagg tgaagatcct ttttgataat ctcatgccat cgtgccgcca
     3421 gaaattgtaa acgttaatat tttgttaaaa ttcgcgttaa atttttgtta aatcagctca
     3481 ttttttaacc aataggccga aatcggcaaa atcccttata aatcaaaaga atagaccgag
     3541 atagggttga gtgttgttcc agtttggaac aagagtccac tattaaagaa cgtggactcc
     3601 aacgtcaaag ggcgaaaaac cgtctatcag ggcgatggcc cactacgtga accatcaccc
     3661 taatcaagtt ttttggggtc gaggtgccgt aaagcactaa atcggaaccc taaagggagc
     3721 ccccgattta gagcttgacg gggaaagccg gcgaacgtgg cgagaaagga agggaagaaa
     3781 gcgaaaggag cgggcgctag ggcgctggca agtgtagcgg tcacgctgcg cgtaaccacc
     3841 acacccgccg cgcttaatgc gccgctacag ggcgcgtccc attcgccaat ccggatatag
     3901 ttcctccttt cagcaaaaaa cccctcaaga cccgtttaga ggccccaagg ggttatgcta
     3961 gttattgctc agcggtggca gcagcctagg ttaattaatt tttctcaaac tgagggtgtg
     4021 accatgcact accacctgaa ccaccaccgc taccaccacc tttttcaaac tgcggatggc
     4081 tccagctagc catatggctg ccgcgcggca ccaggccgct gctggattcg ctgataacgg
     4141 tctggcgtgg ctctggagcc ttcagaaagc catagccata gccgtcggaa ttgaagagct
     4201 tggtcatatg ctttgtgtgg cattgtatgc gtttaccagt cttggacttg aacccagcta
     4261 aaggcttcga tgttgtagtt tcggcaatac gactgacgta ggtgccgaca tacttgccct
     4321 tgggaatttc agcccgaaca atgtcgccgg tgcgaaacgg atgaaccgga attcctttgg
     4381 ccttacctct agggaaaccg taagcatcta cgttgacaac ctgccgtttc ccgtgcccca
     4441 tcgccttgat cgctagtggt ctgaccgctt cccactgcag cacttccggt gtactggctc
     4501 cgacacaagc ggcatccagc cagtgtgtct tgggaatctg tcgctcagaa cgattgaact
     4561 tggtgcggcc accgctggca acttccacag gcaagccagt agctttcaga cgttccagta
     4621 gagcgtatcg ggttgagttg acggctgcag catccttcag tggagccttg gcctgacgtt
     4681 ggagtttctg caacacttcc ggtttttttg ccaggaacac ggctgcagga cgttggttct
     4741 ttcttttgtt gcagggttca caagatagcg tcaggttgct gacccgatca ctgccgccgt
     4801 ggctacgtgg aacaatgtga tccacttcca aacgcttgtc cttggcaccg caatagccgc
     4861 actgtcgccg ccacttgtcc aacaggtatt cccgcacctc gtaaccgaat agagtgccct
     4921 gctggtactc cacgccactg atttccgggt tctgcatctt ctgcatatca aagcgcacaa
     4981 tctcttgtga aatcgccgtg atgggcaggt agcgccgcaa gcgatgcacc caggtcatga
     5041 tgacttcaat gcgatgttgg agcgatggag cgagccactt ccccaacgta gaaaccgtgt
     5101 cagcactgga tagcaaaggt gggccagtac gcttgcgatt cagccaacgg gcaggacggt
     5161 agcgggtctt gcggttgcgt cttccacggc gtagcgatcg gcgggattcc agggcattct
     5221 tgatcagctg accacggtgc tggagttctg ccgcccagac cacttcgcca gtggcttctg
     5281 tgactacggc aatgccggta gtcttgctgc ctggatctag tttcagccgc acaggtcgtg
     5341 ggcttgcggt agggtaggat tgtttgagga tgatagtgaa tgggtatcga cgcaacaccg
     5401 ctgccttgcc actgttcagt agcaggcgag cccgtcttgg cgtacaaggc gccagcggac
     5461 aacgatcagc gtcaacaacc aagactctgg aggatccact tgagccacca gtctgatgaa
     5521 gcattgcgtc gatttcgtcg ccatcttcca tctccagttc atccggggtc tgttccgcac
     5581 gcaggcgacg accatcaaac aggaaagcaa ttgcggtcat gtctacagac tgacggtcac
     5641 agtatgcgtt catcagcttt ttcagctggg tagaacgttt gatgcggaaa aagacttcgt
     5701 tgccgtcttg acctttcact ttcaggttga tatgggcacc gccaccttcg ccacctgccg
     5761 gtttcttatc ttcttcgcca cccgcagctg agctcgaatt agtctgcgcg tctttcaggg
     5821 cttcatcgac agtctgacga ccgctggcgg cgttgatcac cgcagtacgc acggcatacc
     5881 agaaagcgga catctgcggg atgttcggca tgatttcacc tttctgggcg ttttccatgg
     5941 tggcggcaat acgtggatct ttcgccaact cttcctcgta agacttcagc gctacggcac
     6001 ccagcggttt gtctttatta accgcttcca gaccttcatc agtcagcaga tagttttcga
     6061 ggaactcttt tgccagctct ttgttcggac tggcggcgtt aatacctgcg ctcagcacgc
     6121 caacgaacgg tttggatggt tgacccttga aggtcggcag taccgttaca ccataattca
     6181 ctttgctggt gtcgatgttg gaccatgccc acgggccgtt gatggtcatc gctgtttcgc
     6241 ctttattaaa ggcagcttct gcgatggagt aatcagtgtc tgcattcatg tgtttgtttt
     6301 taatcaggtc aaccaggaag gtcagacccg ctttcgcgcc agcgttatcc acgcccacgt
     6361 ctttaatgtc gtacttgccg ttttcatact tgaacgcata acccccgtca gcagcaatca
     6421 gcggccaggt gaagtacggt tcttgcaggt tgaacatcag cgcgctctta cctttcgctt
     6481 tcagttcttt atccagcgcc gggatctctt cccaggtttt tggcgggttc ggcagcagat
     6541 ctttgttata aatcagcgat aacgcttcaa cagcgatcgg gtaagcaatc agcttgccgt
     6601 tgtaacgtac ggcatcccag gtaaacggat acagcttgtc ctggaacgct ttgtccgggg
     6661 tgatttcagc caacaggcca gattgagcgt agccaccaaa gcggtcgtgt gcccagaaga
     6721 taatgtcagg gccatcgcca gttgccgcaa cctgtgggaa tttctcttcc agtttatccg
     6781 gatgctcaac ggtgacttta attccggtat ctttctcgaa tttcttaccg acttcagcga
     6841 gaccgttata gcctttatcg ccgttaatcc agattaccag tttaccttct tcagttttcc
     6901 cggtatggtg atgactgcct gaatgatggt ggtgtccggt atggtgatgg cctgaatgat
     6961 ggtgatgctt gctcatggta tatctccttc ttaaagttaa acaaaattat ttctagaggg
     7021 gaattgttat ccgctcacaa ttcccctata gtgagtcgta ttaatttcgc gggatcgaga
     7081 tcgatctcga tcctctacgc cggacgcatc gtggccggca tcaccggcgc cacaggtgcg
     7141 gttgctggcg cctatatcgc cgacatcacc gatggggaag atcgggctcg ccacttcggg
     7201 ctcatgagcg cttgtttcgg cgtgggtatg gtggcaggcc ccgtggccgg gggactgttg
     7261 ggcgccatct ccttgcatgc accattcctt gcggcggcgg tgctcaacgg cctcaaccta
     7321 ctactgggct gcttcctaat gcaggagtcg cataagggag agcgtcgaga tcccggacac
     7381 catcgaatgg cgcaaaacct ttcgcggtat ggcatgatag cgcccggaag agagtcaatt
     7441 cagggtggtg aatgtgaaac cagtaacgtt atacgatgtc gcagagtatg ccggtgtctc
     7501 ttatcagacc gtttcccgcg tggtgaacca ggccagccac gtttctgcga aaacgcggga
     7561 aaaagtggaa gcggcgatgg cggagctgaa ttacattccc aaccgcgtgg cacaacaact
     7621 ggcgggcaaa cagtcgttgc tgattggcgt tgccacctcc agtctggccc tgcacgcgcc
     7681 gtcgcaaatt gtcgcggcga ttaaatctcg cgccgatcaa ctgggtgcca gcgtggtggt
     7741 gtcgatggta gaacgaagcg gcgtcgaagc ctgtaaagcg gcggtgcaca atcttctcgc
     7801 gcaacgcgtc agtgggctga tcattaacta tccgctggat gaccaggatg ccattgctgt
     7861 ggaagctgcc tgcactaatg ttccggcgtt atttcttgat gtctctgacc agacacccat
     7921 caacagtatt attttctccc atgaagacgg tacgcgactg ggcgtggagc atctggtcgc
     7981 attgggtcac cagcaaatcg cgctgttagc gggcccatta agttctgtct cggcgcgtct
     8041 g
//
