LOCUS       sequence-266857-        6584 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression construct for ShTniQ.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6584)
  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 6584)
  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: pXT131_TwinStrep-SUMO-ShTniQ
FEATURES             Location/Qualifiers
     source          1..6584
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             81..98
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             108..125
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(138..161,162..197,198..221)
                     /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             222..515
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(1066..1084)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1080..1127
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1302..1307,1308..1324,1325..1330))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1357..1375)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1443..1547
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1548..1616,1617..2408)
                     /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(1766..1785)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2579..3167
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3068..3087
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3321..3338
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3353..3492
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3418..3437)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3578..3600
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(3594..3785)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5097..6179)
                     /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     6141..6160
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6180..6257)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     6463..6482
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(6566..6584,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        6566..6584
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatataccat gggcagcagc catcatcatc atcatcacag cagcggcctg gtgccgcgcg
      121 gcagccatat ggctagctgg agccatccgc agtttgaaaa aggtggtggt agcggtggtg
      181 gttcaggtgg tagtgcatgg tcacaccctc agtttgagaa aatgtcggac tcagaagtca
      241 atcaagaagc taagccagag gtcaagccag aagtcaagcc tgagactcac atcaatttaa
      301 aggtgtccga tggatcttca gagatcttct tcaagatcaa aaagaccact cctttaagaa
      361 ggctgatgga agcgttcgct aaaagacagg gtaaggaaat ggactcctta agattcttgt
      421 acgacggtat tagaatccaa gctgatcaga cccctgaaga tttggacatg gaggataacg
      481 atattattga ggctcacaga gaacagattg gtggatccat cgaggcccct gacgtgaagc
      541 cttggctgtt cctgatcaag ccctacgagg gcgagagcct gtctcacttc ctgggcagat
      601 tcagacgggc caatcacctg tctgcctctg gcctgggaac actggctgga atcggagcca
      661 ttgtggccag atgggagaga ttccacttca accccagacc tagccagcaa gagctggaag
      721 ccattgccag cgtggtggaa gtggatgccc agagactggc tcagatgctt cctcctgctg
      781 gcgtgggaat gcagcacgag cctattagac tgtgcggcgc ctgttacgcc gagtctcctt
      841 gtcacagaat cgagtggcag tacaagtccg tgtggaagtg cgaccggcac cagctgaaga
      901 tcctggccaa gtgtcccaac tgtcaggccc ctttcaagat gcccgctctg tgggaggatg
      961 gctgctgcca cagatgcaga atgccctttg ccgagatggc caagctgcag aaagtgtgag
     1021 cggccgcact cgaggcccga aaggaagctg agttggctgc tgccaccgct gagcaataac
     1081 tagcataacc ccttggggcc tctaaacggg tcttgagggg ttttttgctg aaaggaggaa
     1141 ctatatccgg atatcccgca agaggcccgg cagtaccggc ataaccaagc ctatgcctac
     1201 agcatccagg gtgacggtgc cgaggatgac gatgagcgca ttgttagatt tcatacacgg
     1261 tgcctgactg cgttagcaat ttaactgtga taaactaccg cattaaagct tatcgatgat
     1321 aagctgtcaa acatgagaat tcttgaagac gaaagggcct cgtgatacgc ctatttttat
     1381 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg
     1441 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga
     1501 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac
     1561 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc
     1621 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca
     1681 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc
     1741 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt gttgacgccg
     1801 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac
     1861 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca
     1921 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg
     1981 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac
     2041 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gcagcaatgg
     2101 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat
     2161 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg
     2221 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg
     2281 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc
     2341 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc
     2401 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt
     2461 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt
     2521 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     2581 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     2641 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     2701 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     2761 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     2821 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     2881 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     2941 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     3001 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     3061 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     3121 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     3181 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     3241 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     3301 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     3361 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     3421 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     3481 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     3541 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     3601 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     3661 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     3721 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     3781 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     3841 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     3901 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     3961 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     4021 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     4081 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     4141 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     4201 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac
     4261 ccaacgctgc ccgagatgcg ccgcgtgcgg ctgctggaga tggcggacgc gatggatatg
     4321 ttctgccaag ggttggtttg cgcattcaca gttctccgca agaattgatt ggctccaatt
     4381 cttggagtgg tgaatccgtt agcgaggtgc cgccggcttc cattcaggtc gaggtggccc
     4441 ggctccatgc accgcgacgc aacgcgggga ggcagacaag gtatagggcg gcgcctacaa
     4501 tccatgccaa cccgttccat gtgctcgccg aggcggcata aatcgccgtg acgatcagcg
     4561 gtccaatgat cgaagttagg ctggtaagag ccgcgagcga tccttgaagc tgtccctgat
     4621 ggtcgtcatc tacctgcctg gacagcatgg cctgcaacgc gggcatcccg atgccgccgg
     4681 aagcgagaag aatcataatg gggaaggcca tccagcctcg cgtcgcgaac gccagcaaga
     4741 cgtagcccag cgcgtcggcc gccatgccgg cgataatggc ctgcttctcg ccgaaacgtt
     4801 tggtggcggg accagtgacg aaggcttgag cgagggcgtg caagattccg aataccgcaa
     4861 gcgacaggcc gatcatcgtc gcgctccagc gaaagcggtc ctcgccgaaa atgacccaga
     4921 gcgctgccgg cacctgtcct acgagttgca tgataaagaa gacagtcata agtgcggcga
     4981 cgatagtcat gccccgcgcc caccggaagg agctgactgg gttgaaggct ctcaagggca
     5041 tcggtcgaga tcccggtgcc taatgagtga gctaacttac attaattgcg ttgcgctcac
     5101 tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg
     5161 cggggagagg cggtttgcgt attgggcgcc agggtggttt ttcttttcac cagtgagacg
     5221 ggcaacagct gattgccctt caccgcctgg ccctgagaga gttgcagcaa gcggtccacg
     5281 ctggtttgcc ccagcaggcg aaaatcctgt ttgatggtgg ttaacggcgg gatataacat
     5341 gagctgtctt cggtatcgtc gtatcccact accgagatat ccgcaccaac gcgcagcccg
     5401 gactcggtaa tggcgcgcat tgcgcccagc gccatctgat cgttggcaac cagcatcgca
     5461 gtgggaacga tgccctcatt cagcatttgc atggtttgtt gaaaaccgga catggcactc
     5521 cagtcgcctt cccgttccgc tatcggctga atttgattgc gagtgagata tttatgccag
     5581 ccagccagac gcagacgcgc cgagacagaa cttaatgggc ccgctaacag cgcgatttgc
     5641 tggtgaccca atgcgaccag atgctccacg cccagtcgcg taccgtcttc atgggagaaa
     5701 ataatactgt tgatgggtgt ctggtcagag acatcaagaa ataacgccgg aacattagtg
     5761 caggcagctt ccacagcaat ggcatcctgg tcatccagcg gatagttaat gatcagccca
     5821 ctgacgcgtt gcgcgagaag attgtgcacc gccgctttac aggcttcgac gccgcttcgt
     5881 tctaccatcg acaccaccac gctggcaccc agttgatcgg cgcgagattt aatcgccgcg
     5941 acaatttgcg acggcgcgtg cagggccaga ctggaggtgg caacgccaat cagcaacgac
     6001 tgtttgcccg ccagttgttg tgccacgcgg ttgggaatgt aattcagctc cgccatcgcc
     6061 gcttccactt tttcccgcgt tttcgcagaa acgtggctgg cctggttcac cacgcgggaa
     6121 acggtctgat aagagacacc ggcatactct gcgacatcgt ataacgttac tggtttcaca
     6181 ttcaccaccc tgaattgact ctcttccggg cgctatcatg ccataccgcg aaaggttttg
     6241 cgccattcga tggtgtccgg gatctcgacg ctctccctta tgcgactcct gcattaggaa
     6301 gcagcccagt agtaggttga ggccgttgag caccgccgcc gcaaggaatg gtgcatgcaa
     6361 ggagatggcg cccaacagtc ccccggccac ggggcctgcc accataccca cgccgaaaca
     6421 agcgctcatg agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata
     6481 ggcgccagca accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag
     6541 gatcgagatc tcgatcccgc gaaattaata cgactcacta tagg
//
