LOCUS       sequence-330726-        7469 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PmcTnsD expression construct for protein purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7469)
  AUTHORS   Saito M, Ladha A, Strecker J, Faure G, Neumann E, Altae-Tran H, 
            Macrae RK, Zhang F
  TITLE     Dual modes of CRISPR-associated transposon homing.
  JOURNAL   Cell. 2021 Apr 29;184(9):2441-2453.e18. doi: 
            10.1016/j.cell.2021.03.006. Epub 2021 Mar 25.
  PUBMED    33770501
REFERENCE   2  (bases 1 to 7469)
  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: 
            "10.1016/j.cell.2021.03.006"; journalName: "Cell"; date: 
            "2021-04-29- 29"; volume: "184"; issue: "9"; pages: "2441-2453.e18"
COMMENT     Sequence Label: pMS33: pTwinStrep-SUMO-PmcTnsD
FEATURES             Location/Qualifiers
     source          1..7469
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             1..294
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(1730..1748)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1744..1791
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1966..1971,1972..1988,1989..1994))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2021..2039)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2107..2211
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2212..2280,2281..3072)
                     /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(2430..2449)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3243..3831
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3732..3751
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3985..4002
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4017..4156
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4082..4101)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4242..4264
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4258..4449)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5761..6843)
                     /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     6805..6824
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6844..6921)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7127..7146
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     7230..7249
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7230..7248
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    7249..7273
                     /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             7288..7310
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             7329..7346
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             7356..7373
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(7386..7409,7410..7445,7446..7469)
                     /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"
ORIGIN
        1 atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct
       61 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa
      121 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg
      181 gactccttaa gattcttgta cgacggtatt agaatccaag ctgatcagac ccctgaagat
      241 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggatcctta
      301 gctagtgtat tagaaacata cgaatcttgg aatttaaaaa agccattaat tccacaacgt
      361 agtcgcctct atcaacctca acctgtaggt attgggactc cttacataga gagtttaaca
      421 ggttatatca cacggatagc agagttgcat ggagttcttc caggagtctt gatgactaga
      481 gaaattgctc cgcttgtcaa taaaatatat ttccaaaatg gagctaatcg aggttttcgt
      541 gaaatattca accgtagtca agcgttaaat ggcatgggag agatggcagc agatttagtt
      601 caagtgttgc aaaaattgac gcttagagat gacttgcgtt ttttaaccat gctgttttgg
      661 tctaacattt tgactccacg caatttattt cgtaccagaa aagcttggtg tccaatttgt
      721 tatcaagagc ggcatcaaaa tgggctggta gtctatgagc aattgctttg gacaattaac
      781 ctaattacaa tttgcccgca gcatcaaaaa cccttggttg aactttgccc tcactgtaat
      841 catgaatcac ctttgttaaa ctggcgctca agacctggtt actgttcaaa gtgtggagag
      901 tggttaggcg caaatcaatg ccttaagaca tttacagatg gtgaaggatc aataaaatta
      961 cagttggagt ggcagtattg gactgccaat gttgtaggag agctaatttt agcttctcaa
     1021 tgctttgaat ctgccccatc aaaagaaaat attacaaaat cattaaatat agttattgat
     1081 aaagttgcgg aaaataatgc agctgcattt tctcgtctta ttggagtacc aaaaaatagt
     1141 ttgtggatgt ggcagtctac taaaaccttg ccagagctaa atacacttct gaaaatttgt
     1201 tacgaattag agatatcgtt ggtagaattt cttactccaa aaaatctaat tactaaatct
     1261 tttacaaaaa tctcgcagaa gcacctccaa ctttctcgca caccaagggt atctccaaaa
     1321 tctttcgacc aatatcaagt aaaagatgct ttattagcaa tacttgcagg taatgaagaa
     1381 ccaccaccaa caatggaaga agttggaaag cgtttaggtc atcacaacag aactatttct
     1441 agacactttc cagatttgtg tagtgctatt tctgctaaat gtcgtaatta caataaagct
     1501 tgccgtctca aaagcataga aaaactttgc agcgaagtta gagagattgt ccttagcctt
     1561 aatgctcagg gtgtgtatcc cacagaaggt cgtgtttgtg aattaatgcc aaatccgggg
     1621 tgttttagat ataaacaagt ccgcgctgct tttaatgatg cacgacgtga gtttggtttg
     1681 taagcggccg cactcgaggc ccgaaaggaa gctgagttgg ctgctgccac cgctgagcaa
     1741 taactagcat aaccccttgg ggcctctaaa cgggtcttga ggggtttttt gctgaaagga
     1801 ggaactatat ccggatatcc cgcaagaggc ccggcagtac cggcataacc aagcctatgc
     1861 ctacagcatc cagggtgacg gtgccgagga tgacgatgag cgcattgtta gatttcatac
     1921 acggtgcctg actgcgttag caatttaact gtgataaact accgcattaa agcttatcga
     1981 tgataagctg tcaaacatga gaattcttga agacgaaagg gcctcgtgat acgcctattt
     2041 ttataggtta atgtcatgat aataatggtt tcttagacgt caggtggcac ttttcgggga
     2101 aatgtgcgcg gaacccctat ttgtttattt ttctaaatac attcaaatat gtatccgctc
     2161 atgagacaat aaccctgata aatgcttcaa taatattgaa aaaggaagag tatgagtatt
     2221 caacatttcc gtgtcgccct tattcccttt tttgcggcat tttgccttcc tgtttttgct
     2281 cacccagaaa cgctggtgaa agtaaaagat gctgaagatc agttgggtgc acgagtgggt
     2341 tacatcgaac tggatctcaa cagcggtaag atccttgaga gttttcgccc cgaagaacgt
     2401 tttccaatga tgagcacttt taaagttctg ctatgtggcg cggtattatc ccgtgttgac
     2461 gccgggcaag agcaactcgg tcgccgcata cactattctc agaatgactt ggttgagtac
     2521 tcaccagtca cagaaaagca tcttacggat ggcatgacag taagagaatt atgcagtgct
     2581 gccataacca tgagtgataa cactgcggcc aacttacttc tgacaacgat cggaggaccg
     2641 aaggagctaa ccgctttttt gcacaacatg ggggatcatg taactcgcct tgatcgttgg
     2701 gaaccggagc tgaatgaagc cataccaaac gacgagcgtg acaccacgat gcctgcagca
     2761 atggcaacaa cgttgcgcaa actattaact ggcgaactac ttactctagc ttcccggcaa
     2821 caattaatag actggatgga ggcggataaa gttgcaggac cacttctgcg ctcggccctt
     2881 ccggctggct ggtttattgc tgataaatct ggagccggtg agcgtgggtc tcgcggtatc
     2941 attgcagcac tggggccaga tggtaagccc tcccgtatcg tagttatcta cacgacgggg
     3001 agtcaggcaa ctatggatga acgaaataga cagatcgctg agataggtgc ctcactgatt
     3061 aagcattggt aactgtcaga ccaagtttac tcatatatac tttagattga tttaaaactt
     3121 catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat gaccaaaatc
     3181 ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat caaaggatct
     3241 tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa accaccgcta
     3301 ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa ggtaactggc
     3361 ttcagcagag cgcagatacc aaatactgtc cttctagtgt agccgtagtt aggccaccac
     3421 ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt accagtggct
     3481 gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata gttaccggat
     3541 aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt ggagcgaacg
     3601 acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac gcttcccgaa
     3661 gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga gcgcacgagg
     3721 gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg ccacctctga
     3781 cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa aaacgccagc
     3841 aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat gttctttcct
     3901 gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc tgataccgct
     3961 cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga agagcgcctg
     4021 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcaatgg tgcactctca
     4081 gtacaatctg ctctgatgcc gcatagttaa gccagtatac actccgctat cgctacgtga
     4141 ctgggtcatg gctgcgcccc gacacccgcc aacacccgct gacgcgccct gacgggcttg
     4201 tctgctcccg gcatccgctt acagacaagc tgtgaccgtc tccgggagct gcatgtgtca
     4261 gaggttttca ccgtcatcac cgaaacgcgc gaggcagctg cggtaaagct catcagcgtg
     4321 gtcgtgaagc gattcacaga tgtctgcctg ttcatccgcg tccagctcgt tgagtttctc
     4381 cagaagcgtt aatgtctggc ttctgataaa gcgggccatg ttaagggcgg ttttttcctg
     4441 tttggtcact gatgcctccg tgtaaggggg atttctgttc atgggggtaa tgataccgat
     4501 gaaacgagag aggatgctca cgatacgggt tactgatgat gaacatgccc ggttactgga
     4561 acgttgtgag ggtaaacaac tggcggtatg gatgcggcgg gaccagagaa aaatcactca
     4621 gggtcaatgc cagcgcttcg ttaatacaga tgtaggtgtt ccacagggta gccagcagca
     4681 tcctgcgatg cagatccgga acataatggt gcagggcgct gacttccgcg tttccagact
     4741 ttacgaaaca cggaaaccga agaccattca tgttgttgct caggtcgcag acgttttgca
     4801 gcagcagtcg cttcacgttc gctcgcgtat cggtgattca ttctgctaac cagtaaggca
     4861 accccgccag cctagccggg tcctcaacga caggagcacg atcatgcgca cccgtggcca
     4921 ggacccaacg ctgcccgaga tgcgccgcgt gcggctgctg gagatggcgg acgcgatgga
     4981 tatgttctgc caagggttgg tttgcgcatt cacagttctc cgcaagaatt gattggctcc
     5041 aattcttgga gtggtgaatc cgttagcgag gtgccgccgg cttccattca ggtcgaggtg
     5101 gcccggctcc atgcaccgcg acgcaacgcg gggaggcaga caaggtatag ggcggcgcct
     5161 acaatccatg ccaacccgtt ccatgtgctc gccgaggcgg cataaatcgc cgtgacgatc
     5221 agcggtccaa tgatcgaagt taggctggta agagccgcga gcgatccttg aagctgtccc
     5281 tgatggtcgt catctacctg cctggacagc atggcctgca acgcgggcat cccgatgccg
     5341 ccggaagcga gaagaatcat aatggggaag gccatccagc ctcgcgtcgc gaacgccagc
     5401 aagacgtagc ccagcgcgtc ggccgccatg ccggcgataa tggcctgctt ctcgccgaaa
     5461 cgtttggtgg cgggaccagt gacgaaggct tgagcgaggg cgtgcaagat tccgaatacc
     5521 gcaagcgaca ggccgatcat cgtcgcgctc cagcgaaagc ggtcctcgcc gaaaatgacc
     5581 cagagcgctg ccggcacctg tcctacgagt tgcatgataa agaagacagt cataagtgcg
     5641 gcgacgatag tcatgccccg cgcccaccgg aaggagctga ctgggttgaa ggctctcaag
     5701 ggcatcggtc gagatcccgg tgcctaatga gtgagctaac ttacattaat tgcgttgcgc
     5761 tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa
     5821 cgcgcgggga gaggcggttt gcgtattggg cgccagggtg gtttttcttt tcaccagtga
     5881 gacgggcaac agctgattgc ccttcaccgc ctggccctga gagagttgca gcaagcggtc
     5941 cacgctggtt tgccccagca ggcgaaaatc ctgtttgatg gtggttaacg gcgggatata
     6001 acatgagctg tcttcggtat cgtcgtatcc cactaccgag atatccgcac caacgcgcag
     6061 cccggactcg gtaatggcgc gcattgcgcc cagcgccatc tgatcgttgg caaccagcat
     6121 cgcagtggga acgatgccct cattcagcat ttgcatggtt tgttgaaaac cggacatggc
     6181 actccagtcg ccttcccgtt ccgctatcgg ctgaatttga ttgcgagtga gatatttatg
     6241 ccagccagcc agacgcagac gcgccgagac agaacttaat gggcccgcta acagcgcgat
     6301 ttgctggtga cccaatgcga ccagatgctc cacgcccagt cgcgtaccgt cttcatggga
     6361 gaaaataata ctgttgatgg gtgtctggtc agagacatca agaaataacg ccggaacatt
     6421 agtgcaggca gcttccacag caatggcatc ctggtcatcc agcggatagt taatgatcag
     6481 cccactgacg cgttgcgcga gaagattgtg caccgccgct ttacaggctt cgacgccgct
     6541 tcgttctacc atcgacacca ccacgctggc acccagttga tcggcgcgag atttaatcgc
     6601 cgcgacaatt tgcgacggcg cgtgcagggc cagactggag gtggcaacgc caatcagcaa
     6661 cgactgtttg cccgccagtt gttgtgccac gcggttggga atgtaattca gctccgccat
     6721 cgccgcttcc actttttccc gcgttttcgc agaaacgtgg ctggcctggt tcaccacgcg
     6781 ggaaacggtc tgataagaga caccggcata ctctgcgaca tcgtataacg ttactggttt
     6841 cacattcacc accctgaatt gactctcttc cgggcgctat catgccatac cgcgaaaggt
     6901 tttgcgccat tcgatggtgt ccgggatctc gacgctctcc cttatgcgac tcctgcatta
     6961 ggaagcagcc cagtagtagg ttgaggccgt tgagcaccgc cgccgcaagg aatggtgcat
     7021 gcaaggagat ggcgcccaac agtcccccgg ccacggggcc tgccaccata cccacgccga
     7081 aacaagcgct catgagcccg aagtggcgag cccgatcttc cccatcggtg atgtcggcga
     7141 tataggcgcc agcaaccgca cctgtggcgc cggtgatgcc ggccacgatg cgtccggcgt
     7201 agaggatcga gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc
     7261 ggataacaat tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg
     7321 gcagcagcca tcatcatcat catcacagca gcggcctggt gccgcgcggc agccatatgg
     7381 ctagctggag ccatccgcag tttgaaaaag gtggtggtag cggtggtggt tcaggtggta
     7441 gtgcatggtc acaccctcag tttgagaaa
//
