LOCUS       sequence-266862-        6900 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression construct for ShTnsC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6900)
  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 6900)
  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: pXT130_TwinStrep-SUMO-ShTnsC
FEATURES             Location/Qualifiers
     source          1..6900
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..18
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             28..45
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(58..81,82..117,118..141)
                     /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             142..435
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(1313..1331)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1327..1374
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1549..1554,1555..1571,1572..1577))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1604..1622)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1690..1794
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1795..1863,1864..2655)
                     /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(2013..2032)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2826..3414
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3315..3334
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3557..3574
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3589..3728
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3654..3673)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3814..3836
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3830..4021)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5333..6415)
                     /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     6377..6396
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6416..6493)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6699..6718
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     6802..6821
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6802..6820
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    6821..6845
                     /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             6860..6882
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
ORIGIN
        1 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatat ggctagctgg
       61 agccatccgc agtttgaaaa aggtggtggt agcggtggtg gttcaggtgg tagtgcatgg
      121 tcacaccctc agtttgagaa aatgtcggac tcagaagtca atcaagaagc taagccagag
      181 gtcaagccag aagtcaagcc tgagactcac atcaatttaa aggtgtccga tggatcttca
      241 gagatcttct tcaagatcaa aaagaccact cctttaagaa ggctgatgga agcgttcgct
      301 aaaagacagg gtaaggaaat ggactcctta agattcttgt acgacggtat tagaatccaa
      361 gctgatcaga cccctgaaga tttggacatg gaggataacg atattattga ggctcacaga
      421 gaacagattg gtggatccac agaggcccag gccattgcca aacagctcgg cggagtgaag
      481 cccgacgatg aatggctgca ggccgagatc gctagactga agggcaagag catcgtgccc
      541 ctgcagcaag tgaaaaccct gcacgattgg ctggacggca agcggaaggc cagaaagtcc
      601 tgtagagtcg tgggcgagag cagaaccgga aagaccgtgg cctgtgacgc ctaccggtac
      661 agacacaagc ctcagcaaga agccggcaga cctcctacag tgcccgtggt gtatatcaga
      721 ccccaccaga agtgcggccc caaggacctg ttcaagaaga tcaccgagta cctgaagtac
      781 cgcgtgacca agggcaccgt gtccgacttc agagacagaa ccatcgaggt gctgaaaggc
      841 tgcggcgtgg aaatgctgat catcgacgag gccgaccggc tgaagcctga gacatttgct
      901 gacgtgcggg atatcgccga ggacctggga attgctgtgg tgctcgtggg caccgataga
      961 ctggatgccg tgatcaagcg ggacgaacag gtgctggaac ggttcagagc ccacctgaga
     1021 ttcggcaagc tgagcggcga ggacttcaag aacacagtgg aaatgtggga gcagatggtg
     1081 ctgaagctgc ccgtgtccag caacctgaag tccaaagaga tgctgcggat cctgaccagc
     1141 gccaccgagg gatatatcgg cagactggac gagatcctga gagaggccgc catcagaagc
     1201 ctgtccagag gcctgaagaa aatcgacaag gccgtgctgc aagaggtggc caaagagtac
     1261 aagtaagcgg ccgcactcga ggcccgaaag gaagctgagt tggctgctgc caccgctgag
     1321 caataactag cataacccct tggggcctct aaacgggtct tgaggggttt tttgctgaaa
     1381 ggaggaacta tatccggata tcccgcaaga ggcccggcag taccggcata accaagccta
     1441 tgcctacagc atccagggtg acggtgccga ggatgacgat gagcgcattg ttagatttca
     1501 tacacggtgc ctgactgcgt tagcaattta actgtgataa actaccgcat taaagcttat
     1561 cgatgataag ctgtcaaaca tgagaattct tgaagacgaa agggcctcgt gatacgccta
     1621 tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg cacttttcgg
     1681 ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg
     1741 ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtatgagt
     1801 attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct tcctgttttt
     1861 gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg
     1921 ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg ccccgaagaa
     1981 cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt atcccgtgtt
     2041 gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga cttggttgag
     2101 tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga attatgcagt
     2161 gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac gatcggagga
     2221 ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg ccttgatcgt
     2281 tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac gatgcctgca
     2341 gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct agcttcccgg
     2401 caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct gcgctcggcc
     2461 cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt
     2521 atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat ctacacgacg
     2581 gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg tgcctcactg
     2641 attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat tgatttaaaa
     2701 cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa
     2761 atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga
     2821 tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg
     2881 ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact
     2941 ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac
     3001 cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg
     3061 gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg
     3121 gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga
     3181 acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc
     3241 gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg
     3301 agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc
     3361 tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc
     3421 agcaacgcgg cctttttacg gttgctggcc ttttgctcac atgttctttc ctgcgttatc
     3481 ccctgattct gtggataacc gtattaccgc ctttgagtga gctgataccg ctcgccgcag
     3541 ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta
     3601 ttttctcctt acgcatctgt gcggtatttc acaccgcaat ggtgcactct cagtacaatc
     3661 tgctctgatg ccgcatagtt aagccagtat acactccgct atcgctacgt gactgggtca
     3721 tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc
     3781 cggcatccgc ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt
     3841 caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa
     3901 gcgattcaca gatgtctgcc tgttcatccg cgtccagctc gttgagtttc tccagaagcg
     3961 ttaatgtctg gcttctgata aagcgggcca tgttaagggc ggttttttcc tgtttggtca
     4021 ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt aatgataccg atgaaacgag
     4081 agaggatgct cacgatacgg gttactgatg atgaacatgc ccggttactg gaacgttgtg
     4141 agggtaaaca actggcggta tggatgcggc gggaccagag aaaaatcact cagggtcaat
     4201 gccagcgctt cgttaataca gatgtaggtg ttccacaggg tagccagcag catcctgcga
     4261 tgcagatccg gaacataatg gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa
     4321 cacggaaacc gaagaccatt catgttgttg ctcaggtcgc agacgttttg cagcagcagt
     4381 cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta accagtaagg caaccccgcc
     4441 agcctagccg ggtcctcaac gacaggagca cgatcatgcg cacccgtggc caggacccaa
     4501 cgctgcccga gatgcgccgc gtgcggctgc tggagatggc ggacgcgatg gatatgttct
     4561 gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa ttgattggct ccaattcttg
     4621 gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt caggtcgagg tggcccggct
     4681 ccatgcaccg cgacgcaacg cggggaggca gacaaggtat agggcggcgc ctacaatcca
     4741 tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc
     4801 aatgatcgaa gttaggctgg taagagccgc gagcgatcct tgaagctgtc cctgatggtc
     4861 gtcatctacc tgcctggaca gcatggcctg caacgcgggc atcccgatgc cgccggaagc
     4921 gagaagaatc ataatgggga aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta
     4981 gcccagcgcg tcggccgcca tgccggcgat aatggcctgc ttctcgccga aacgtttggt
     5041 ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag attccgaata ccgcaagcga
     5101 caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc
     5161 tgccggcacc tgtcctacga gttgcatgat aaagaagaca gtcataagtg cggcgacgat
     5221 agtcatgccc cgcgcccacc ggaaggagct gactgggttg aaggctctca agggcatcgg
     5281 tcgagatccc ggtgcctaat gagtgagcta acttacatta attgcgttgc gctcactgcc
     5341 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     5401 gagaggcggt ttgcgtattg ggcgccaggg tggtttttct tttcaccagt gagacgggca
     5461 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
     5521 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
     5581 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
     5641 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
     5701 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
     5761 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
     5821 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
     5881 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
     5941 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
     6001 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
     6061 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
     6121 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
     6181 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
     6241 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
     6301 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
     6361 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
     6421 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     6481 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     6541 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     6601 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     6661 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     6721 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     6781 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     6841 attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat gggcagcagc
//
