LOCUS       sequence-261590-        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"
     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(1562..1580)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1576..1623
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1798..1803,1804..1820,1821..1826))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(1853..1871)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1939..2043
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2044..2112,2113..2904)
                     /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(2262..2281)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3075..3663
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3564..3583
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3817..3834
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    3849..3988
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(3914..3933)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4074..4096
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4090..4281)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(5593..6584,1..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 atccatcgag
     1021 gcccctgacg tgaagccttg gctgttcctg atcaagccct acgagggcga gagcctgtct
     1081 cacttcctgg gcagattcag acgggccaat cacctgtctg cctctggcct gggaacactg
     1141 gctggaatcg gagccattgt ggccagatgg gagagattcc acttcaaccc cagacctagc
     1201 cagcaagagc tggaagccat tgccagcgtg gtggaagtgg atgcccagag actggctcag
     1261 atgcttcctc ctgctggcgt gggaatgcag cacgagccta ttagactgtg cggcgcctgt
     1321 tacgccgagt ctccttgtca cagaatcgag tggcagtaca agtccgtgtg gaagtgcgac
     1381 cggcaccagc tgaagatcct ggccaagtgt cccaactgtc aggccccttt caagatgccc
     1441 gctctgtggg aggatggctg ctgccacaga tgcagaatgc cctttgccga gatggccaag
     1501 ctgcagaaag tgtgagcggc cgcactcgag gcccgaaagg aagctgagtt ggctgctgcc
     1561 accgctgagc aataactagc ataacccctt ggggcctcta aacgggtctt gaggggtttt
     1621 ttgctgaaag gaggaactat atccggatat cccgcaagag gcccggcagt accggcataa
     1681 ccaagcctat gcctacagca tccagggtga cggtgccgag gatgacgatg agcgcattgt
     1741 tagatttcat acacggtgcc tgactgcgtt agcaatttaa ctgtgataaa ctaccgcatt
     1801 aaagcttatc gatgataagc tgtcaaacat gagaattctt gaagacgaaa gggcctcgtg
     1861 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     1921 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     1981 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     2041 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     2101 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     2161 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     2221 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     2281 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     2341 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     2401 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     2461 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     2521 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     2581 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     2641 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     2701 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     2761 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     2821 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     2881 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     2941 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     3001 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     3061 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3121 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3181 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     3241 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3301 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3361 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3421 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3481 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3541 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3601 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3661 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3721 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3781 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     3841 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcaat
     3901 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagtat acactccgct
     3961 atcgctacgt gactgggtca tggctgcgcc ccgacacccg ccaacacccg ctgacgcgcc
     4021 ctgacgggct tgtctgctcc cggcatccgc ttacagacaa gctgtgaccg tctccgggag
     4081 ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc gcgaggcagc tgcggtaaag
     4141 ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc tgttcatccg cgtccagctc
     4201 gttgagtttc tccagaagcg ttaatgtctg gcttctgata aagcgggcca tgttaagggc
     4261 ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg ggatttctgt tcatgggggt
     4321 aatgataccg atgaaacgag agaggatgct cacgatacgg gttactgatg atgaacatgc
     4381 ccggttactg gaacgttgtg agggtaaaca actggcggta tggatgcggc gggaccagag
     4441 aaaaatcact cagggtcaat gccagcgctt cgttaataca gatgtaggtg ttccacaggg
     4501 tagccagcag catcctgcga tgcagatccg gaacataatg gtgcagggcg ctgacttccg
     4561 cgtttccaga ctttacgaaa cacggaaacc gaagaccatt catgttgttg ctcaggtcgc
     4621 agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt atcggtgatt cattctgcta
     4681 accagtaagg caaccccgcc agcctagccg ggtcctcaac gacaggagca cgatcatgcg
     4741 cacccgtggc caggacccaa cgctgcccga gatgcgccgc gtgcggctgc tggagatggc
     4801 ggacgcgatg gatatgttct gccaagggtt ggtttgcgca ttcacagttc tccgcaagaa
     4861 ttgattggct ccaattcttg gagtggtgaa tccgttagcg aggtgccgcc ggcttccatt
     4921 caggtcgagg tggcccggct ccatgcaccg cgacgcaacg cggggaggca gacaaggtat
     4981 agggcggcgc ctacaatcca tgccaacccg ttccatgtgc tcgccgaggc ggcataaatc
     5041 gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg taagagccgc gagcgatcct
     5101 tgaagctgtc cctgatggtc gtcatctacc tgcctggaca gcatggcctg caacgcgggc
     5161 atcccgatgc cgccggaagc gagaagaatc ataatgggga aggccatcca gcctcgcgtc
     5221 gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca tgccggcgat aatggcctgc
     5281 ttctcgccga aacgtttggt ggcgggacca gtgacgaagg cttgagcgag ggcgtgcaag
     5341 attccgaata ccgcaagcga caggccgatc atcgtcgcgc tccagcgaaa gcggtcctcg
     5401 ccgaaaatga cccagagcgc tgccggcacc tgtcctacga gttgcatgat aaagaagaca
     5461 gtcataagtg cggcgacgat agtcatgccc cgcgcccacc ggaaggagct gactgggttg
     5521 aaggctctca agggcatcgg tcgagatccc ggtgcctaat gagtgagcta acttacatta
     5581 attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa
     5641 tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgccaggg tggtttttct
     5701 tttcaccagt gagacgggca acagctgatt gcccttcacc gcctggccct gagagagttg
     5761 cagcaagcgg tccacgctgg tttgccccag caggcgaaaa tcctgtttga tggtggttaa
     5821 cggcgggata taacatgagc tgtcttcggt atcgtcgtat cccactaccg agatatccgc
     5881 accaacgcgc agcccggact cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt
     5941 ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa
     6001 accggacatg gcactccagt cgccttcccg ttccgctatc ggctgaattt gattgcgagt
     6061 gagatattta tgccagccag ccagacgcag acgcgccgag acagaactta atgggcccgc
     6121 taacagcgcg atttgctggt gacccaatgc gaccagatgc tccacgccca gtcgcgtacc
     6181 gtcttcatgg gagaaaataa tactgttgat gggtgtctgg tcagagacat caagaaataa
     6241 cgccggaaca ttagtgcagg cagcttccac agcaatggca tcctggtcat ccagcggata
     6301 gttaatgatc agcccactga cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc
     6361 ttcgacgccg cttcgttcta ccatcgacac caccacgctg gcacccagtt gatcggcgcg
     6421 agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac
     6481 gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt
     6541 cagctccgcc atcgccgctt ccactttttc ccgcgttttc gcag
//
