LOCUS       sequence-261595-        7835 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression construct for ShTnsB.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7835)
  AUTHORS   Strecker J, Ladha A, Gardner Z, Schmid-Burgk JL, Makarova KS, Koonin
            EV, Zhang F
  TITLE     RNA-guided DNA insertion with CRISPR-associated transposases.
  JOURNAL   Science. 2019 Jul 5;365(6448):48-53. doi: 10.1126/science.aax9181. 
            Epub 2019 Jun 6.
  PUBMED    31171706
REFERENCE   2  (bases 1 to 7835)
  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.aax9181"; journalName: "Science"; date: "2019-07-5-
            5"; volume: "365"; issue: "6448"; pages: "48-53"
COMMENT     Sequence Label: pXT129_TwinStrep-SUMO-ShTnsB
FEATURES             Location/Qualifiers
     source          1..7835
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     53..72
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(92..169)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     375..394
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     478..497
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        478..496
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    497..521
                     /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             536..558
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             577..594
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             604..621
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(634..657,658..693,694..717)
                     /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             718..1011
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(2813..2831)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2827..2874
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3049..3054,3055..3071,3072..3077))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3104..3122)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3190..3294
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3295..3363,3364..4155)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(3513..3532)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4326..4914
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4815..4834
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5068..5085
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5100..5239
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5165..5184)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5325..5347
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5341..5532)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6844..7835,1..91))
                     /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"
ORIGIN
        1 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
       61 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
      121 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
      181 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
      241 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
      301 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
      361 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
      421 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
      481 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
      541 ttaactttaa gaaggagata taccatgggc agcagccatc atcatcatca tcacagcagc
      601 ggcctggtgc cgcgcggcag ccatatggct agctggagcc atccgcagtt tgaaaaaggt
      661 ggtggtagcg gtggtggttc aggtggtagt gcatggtcac accctcagtt tgagaaaatg
      721 tcggactcag aagtcaatca agaagctaag ccagaggtca agccagaagt caagcctgag
      781 actcacatca atttaaaggt gtccgatgga tcttcagaga tcttcttcaa gatcaaaaag
      841 accactcctt taagaaggct gatggaagcg ttcgctaaaa gacagggtaa ggaaatggac
      901 tccttaagat tcttgtacga cggtattaga atccaagctg atcagacccc tgaagatttg
      961 gacatggagg ataacgatat tattgaggct cacagagaac agattggtgg atccaacagc
     1021 cagcagaacc ccgatctggc cgtgcatcct ctggctatcc ctatggaagg actgctgggc
     1081 gagagcgcca ccacactgga aaagaacgtg atcgccacac agctgagcga agaggcccaa
     1141 gtgaagctgg aagtgatcca gagcctgctg gaaccctgcg acagaaccac atacggccag
     1201 aagctgagag aggccgccga gaagctgaac gtgtccctga gaacagtgca gcggctggtc
     1261 aagaactggg agcaagatgg actcgtgggc ctgacacaga ccagcagagc cgataagggc
     1321 aagcacagaa tcggcgagtt ctgggagaac ttcatcacca agacctacaa agagggcaac
     1381 aagggcagca agcggatgac ccctaaacag gtggccctga gagtggaagc caaggccaga
     1441 gaactgaagg acagcaagcc tcctaactac aagaccgtgc tgagagtgct ggcccctatc
     1501 ctggaaaagc agcagaaggc caagagcatc agaagccctg gctggcgggg aacaaccctg
     1561 agcgtgaaaa ccagagaggg caaagacctg tccgtggact acagcaacca cgtgtggcag
     1621 tgcgaccaca ccagagtgga tgtgctgctg gtggatcagc acggcgagat cctgtctaga
     1681 ccttggctga ccaccgtgat cgacacctac agcagatgca tcatgggcat caacctgggc
     1741 ttcgacgccc cttctagcgg agttgttgct ctggctctgc ggcacgccat cctgcctaag
     1801 agatacggca gcgagtacaa gctgcactgc gagtggggca cctatggcaa gcctgagcac
     1861 ttctacaccg acggcggcaa ggacttcaga agcaaccacc tgtctcagat cggcgcacag
     1921 ctgggctttg tgtgccacct gagagacaga cctagcgaag gcggcgtggt ggaaagaccc
     1981 ttcaagaccc tgaacgacca gctgttcagc accctgcctg gctacacagg cagcaacgtg
     2041 caagaaagac ccgaggatgc cgagaaggac gccagactga cactgagaga gctggaacag
     2101 ctgcttgtgc ggtacatcgt ggaccggtac aaccagagca tcgacgccag aatgggcgat
     2161 cagaccagat tcgagagatg ggaggccgga ctgcctacag tgcctgtgcc tattcctgag
     2221 cgcgacctgg acatctgcct gatgaagcag agcagacgga cagtgcagag aggcggctgt
     2281 ctgcagttcc agaacctgat gtaccggggc gagtacctgg ctggatatgc cggcgagaca
     2341 gtgaacctga gattcgaccc cagagacatc accaccatcc tggtgtaccg gcaagagaac
     2401 aatcaagagg tgttcctgac cagggctcac gcccagggac tcgaaacaga acaactggcc
     2461 ctggatgaag ccgaagccgc ttctcggaga ctgagaaccg ccggcaagac catcagcaat
     2521 cagtccctgc tgcaagaggt ggtggacaga gatgccctgg tggccactaa gaagtcccgg
     2581 aaagagcggc agaaactgga acagacagtg ctgcgctctg ccgccgtgga cgagagcaat
     2641 agagagagcc tgcctagcca gatcgtggaa cccgatgagg tggaaagcac cgaaaccgtg
     2701 cacagccagt acgaggacat cgaagtgtgg gactacgagc agctgcggga agagtacggc
     2761 ttctgagcgg ccgcactcga ggcccgaaag gaagctgagt tggctgctgc caccgctgag
     2821 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     2881 ggaggaacta tatccggata tcccgcaaga ggcccggcag taccggcata accaagccta
     2941 tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca
     3001 tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat taaagcttat
     3061 cgatgataag ctgtcaaaca tgagaattct tgaagacgaa agggcctcgt gatacgccta
     3121 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     3181 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     3241 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     3301 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     3361 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     3421 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     3481 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     3541 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     3601 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     3661 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     3721 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     3781 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     3841 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     3901 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     3961 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     4021 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     4081 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     4141 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     4201 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     4261 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     4321 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     4381 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     4441 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     4501 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     4561 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     4621 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     4681 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     4741 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     4801 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     4861 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     4921 agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt
     4981 cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc
     5041 gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc
     5101 ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcaa tggtgcactc
     5161 tcagtacaat ctgctctgat gccgcatagt taagccagta tacactccgc tatcgctacg
     5221 tgactgggtc atggctgcgc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc
     5281 ttgtctgctc ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg
     5341 tcagaggttt tcaccgtcat caccgaaacg cgcgaggcag ctgcggtaaa gctcatcagc
     5401 gtggtcgtga agcgattcac agatgtctgc ctgttcatcc gcgtccagct cgttgagttt
     5461 ctccagaagc gttaatgtct ggcttctgat aaagcgggcc atgttaaggg cggttttttc
     5521 ctgtttggtc actgatgcct ccgtgtaagg gggatttctg ttcatggggg taatgatacc
     5581 gatgaaacga gagaggatgc tcacgatacg ggttactgat gatgaacatg cccggttact
     5641 ggaacgttgt gagggtaaac aactggcggt atggatgcgg cgggaccaga gaaaaatcac
     5701 tcagggtcaa tgccagcgct tcgttaatac agatgtaggt gttccacagg gtagccagca
     5761 gcatcctgcg atgcagatcc ggaacataat ggtgcagggc gctgacttcc gcgtttccag
     5821 actttacgaa acacggaaac cgaagaccat tcatgttgtt gctcaggtcg cagacgtttt
     5881 gcagcagcag tcgcttcacg ttcgctcgcg tatcggtgat tcattctgct aaccagtaag
     5941 gcaaccccgc cagcctagcc gggtcctcaa cgacaggagc acgatcatgc gcacccgtgg
     6001 ccaggaccca acgctgcccg agatgcgccg cgtgcggctg ctggagatgg cggacgcgat
     6061 ggatatgttc tgccaagggt tggtttgcgc attcacagtt ctccgcaaga attgattggc
     6121 tccaattctt ggagtggtga atccgttagc gaggtgccgc cggcttccat tcaggtcgag
     6181 gtggcccggc tccatgcacc gcgacgcaac gcggggaggc agacaaggta tagggcggcg
     6241 cctacaatcc atgccaaccc gttccatgtg ctcgccgagg cggcataaat cgccgtgacg
     6301 atcagcggtc caatgatcga agttaggctg gtaagagccg cgagcgatcc ttgaagctgt
     6361 ccctgatggt cgtcatctac ctgcctggac agcatggcct gcaacgcggg catcccgatg
     6421 ccgccggaag cgagaagaat cataatgggg aaggccatcc agcctcgcgt cgcgaacgcc
     6481 agcaagacgt agcccagcgc gtcggccgcc atgccggcga taatggcctg cttctcgccg
     6541 aaacgtttgg tggcgggacc agtgacgaag gcttgagcga gggcgtgcaa gattccgaat
     6601 accgcaagcg acaggccgat catcgtcgcg ctccagcgaa agcggtcctc gccgaaaatg
     6661 acccagagcg ctgccggcac ctgtcctacg agttgcatga taaagaagac agtcataagt
     6721 gcggcgacga tagtcatgcc ccgcgcccac cggaaggagc tgactgggtt gaaggctctc
     6781 aagggcatcg gtcgagatcc cggtgcctaa tgagtgagct aacttacatt aattgcgttg
     6841 cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc
     6901 caacgcgcgg ggagaggcgg tttgcgtatt gggcgccagg gtggtttttc ttttcaccag
     6961 tgagacgggc aacagctgat tgcccttcac cgcctggccc tgagagagtt gcagcaagcg
     7021 gtccacgctg gtttgcccca gcaggcgaaa atcctgtttg atggtggtta acggcgggat
     7081 ataacatgag ctgtcttcgg tatcgtcgta tcccactacc gagatatccg caccaacgcg
     7141 cagcccggac tcggtaatgg cgcgcattgc gcccagcgcc atctgatcgt tggcaaccag
     7201 catcgcagtg ggaacgatgc cctcattcag catttgcatg gtttgttgaa aaccggacat
     7261 ggcactccag tcgccttccc gttccgctat cggctgaatt tgattgcgag tgagatattt
     7321 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
     7381 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
     7441 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
     7501 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
     7561 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
     7621 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
     7681 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
     7741 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
     7801 catcgccgct tccacttttt cccgcgtttt cgcag
//
