LOCUS       sequence-266855-        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"
     RBS             15..37
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             56..73
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             83..100
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(113..136,137..172,173..196)
                     /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             197..490
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(2292..2310)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2306..2353
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2528..2533,2534..2550,2551..2556))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2583..2601)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2669..2773
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2774..2842,2843..3634)
                     /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(2992..3011)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3805..4393
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4294..4313
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4547..4564
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4579..4718
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4644..4663)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4804..4826
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4820..5011)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6323..7405)
                     /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     7367..7386
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7406..7483)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7689..7708
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7792..7811
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7792..7810
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7811..7835
                     /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)."
ORIGIN
        1 cctctagaaa taattttgtt taactttaag aaggagatat accatgggca gcagccatca
       61 tcatcatcat cacagcagcg gcctggtgcc gcgcggcagc catatggcta gctggagcca
      121 tccgcagttt gaaaaaggtg gtggtagcgg tggtggttca ggtggtagtg catggtcaca
      181 ccctcagttt gagaaaatgt cggactcaga agtcaatcaa gaagctaagc cagaggtcaa
      241 gccagaagtc aagcctgaga ctcacatcaa tttaaaggtg tccgatggat cttcagagat
      301 cttcttcaag atcaaaaaga ccactccttt aagaaggctg atggaagcgt tcgctaaaag
      361 acagggtaag gaaatggact ccttaagatt cttgtacgac ggtattagaa tccaagctga
      421 tcagacccct gaagatttgg acatggagga taacgatatt attgaggctc acagagaaca
      481 gattggtgga tccaacagcc agcagaaccc cgatctggcc gtgcatcctc tggctatccc
      541 tatggaagga ctgctgggcg agagcgccac cacactggaa aagaacgtga tcgccacaca
      601 gctgagcgaa gaggcccaag tgaagctgga agtgatccag agcctgctgg aaccctgcga
      661 cagaaccaca tacggccaga agctgagaga ggccgccgag aagctgaacg tgtccctgag
      721 aacagtgcag cggctggtca agaactggga gcaagatgga ctcgtgggcc tgacacagac
      781 cagcagagcc gataagggca agcacagaat cggcgagttc tgggagaact tcatcaccaa
      841 gacctacaaa gagggcaaca agggcagcaa gcggatgacc cctaaacagg tggccctgag
      901 agtggaagcc aaggccagag aactgaagga cagcaagcct cctaactaca agaccgtgct
      961 gagagtgctg gcccctatcc tggaaaagca gcagaaggcc aagagcatca gaagccctgg
     1021 ctggcgggga acaaccctga gcgtgaaaac cagagagggc aaagacctgt ccgtggacta
     1081 cagcaaccac gtgtggcagt gcgaccacac cagagtggat gtgctgctgg tggatcagca
     1141 cggcgagatc ctgtctagac cttggctgac caccgtgatc gacacctaca gcagatgcat
     1201 catgggcatc aacctgggct tcgacgcccc ttctagcgga gttgttgctc tggctctgcg
     1261 gcacgccatc ctgcctaaga gatacggcag cgagtacaag ctgcactgcg agtggggcac
     1321 ctatggcaag cctgagcact tctacaccga cggcggcaag gacttcagaa gcaaccacct
     1381 gtctcagatc ggcgcacagc tgggctttgt gtgccacctg agagacagac ctagcgaagg
     1441 cggcgtggtg gaaagaccct tcaagaccct gaacgaccag ctgttcagca ccctgcctgg
     1501 ctacacaggc agcaacgtgc aagaaagacc cgaggatgcc gagaaggacg ccagactgac
     1561 actgagagag ctggaacagc tgcttgtgcg gtacatcgtg gaccggtaca accagagcat
     1621 cgacgccaga atgggcgatc agaccagatt cgagagatgg gaggccggac tgcctacagt
     1681 gcctgtgcct attcctgagc gcgacctgga catctgcctg atgaagcaga gcagacggac
     1741 agtgcagaga ggcggctgtc tgcagttcca gaacctgatg taccggggcg agtacctggc
     1801 tggatatgcc ggcgagacag tgaacctgag attcgacccc agagacatca ccaccatcct
     1861 ggtgtaccgg caagagaaca atcaagaggt gttcctgacc agggctcacg cccagggact
     1921 cgaaacagaa caactggccc tggatgaagc cgaagccgct tctcggagac tgagaaccgc
     1981 cggcaagacc atcagcaatc agtccctgct gcaagaggtg gtggacagag atgccctggt
     2041 ggccactaag aagtcccgga aagagcggca gaaactggaa cagacagtgc tgcgctctgc
     2101 cgccgtggac gagagcaata gagagagcct gcctagccag atcgtggaac ccgatgaggt
     2161 ggaaagcacc gaaaccgtgc acagccagta cgaggacatc gaagtgtggg actacgagca
     2221 gctgcgggaa gagtacggct tctgagcggc cgcactcgag gcccgaaagg aagctgagtt
     2281 ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt
     2341 gaggggtttt ttgctgaaag gaggaactat atccggatat cccgcaagag gcccggcagt
     2401 accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag gatgacgatg
     2461 agcgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa
     2521 ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaattctt gaagacgaaa
     2581 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     2641 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2701 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2761 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     2821 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     2881 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     2941 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     3001 cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc
     3061 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     3121 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     3181 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     3241 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     3301 tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     3361 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     3421 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     3481 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     3541 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     3601 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     3661 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     3721 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3781 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3841 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3901 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt
     3961 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4021 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4081 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4141 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4201 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4261 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4321 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4381 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4441 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4501 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4561 cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     4621 acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat
     4681 acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg
     4741 ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg
     4801 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc
     4861 tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg
     4921 cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca
     4981 tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt
     5041 tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg
     5101 atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc
     5161 gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg
     5221 ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg
     5281 ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg
     5341 ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt
     5401 cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca
     5461 cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatgcgccgc gtgcggctgc
     5521 tggagatggc ggacgcgatg gatatgttct gccaagggtt ggtttgcgca ttcacagttc
     5581 tccgcaagaa ttgattggct ccaattcttg gagtggtgaa tccgttagcg aggtgccgcc
     5641 ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg cggggaggca
     5701 gacaaggtat agggcggcgc ctacaatcca tgccaacccg ttccatgtgc tcgccgaggc
     5761 ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg taagagccgc
     5821 gagcgatcct tgaagctgtc cctgatggtc gtcatctacc tgcctggaca gcatggcctg
     5881 caacgcgggc atcccgatgc cgccggaagc gagaagaatc ataatgggga aggccatcca
     5941 gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca tgccggcgat
     6001 aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag
     6061 ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa
     6121 gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat
     6181 aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct
     6241 gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta
     6301 acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     6361 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg
     6421 tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct
     6481 gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga
     6541 tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg
     6601 agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca
     6661 tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg
     6721 tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt
     6781 gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta
     6841 atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca
     6901 gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat
     6961 caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat
     7021 ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg
     7081 ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt
     7141 gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg
     7201 aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg
     7261 gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcagaaacgt
     7321 ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca tactctgcga
     7381 catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct tccgggcgct
     7441 atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc tcgacgctct
     7501 cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc gttgagcacc
     7561 gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc ggccacgggg
     7621 cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg agcccgatct
     7681 tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc gccggtgatg
     7741 ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa ttaatacgac
     7801 tcactatagg ggaattgtga gcggataaca attcc
//
