LOCUS       sequence-330724-        8777 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PmcTnsAB expression construct for protein purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8777)
  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 8777)
  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: pMS31: pTwinStrep-SUMO-PmcTnsAB
FEATURES             Location/Qualifiers
     source          1..8777
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     28..47
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     131..150
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        131..149
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    150..174
                     /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             189..211
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             230..247
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             257..274
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(287..310,311..346,347..370)
                     /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             371..664
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(3408..3426)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3422..3469
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3644..3649,3650..3666,3667..3672))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3699..3717)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3785..3889
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3890..3958,3959..4750)
                     /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(4108..4127)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4921..5509
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5410..5429
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5663..5680
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5695..5834
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5760..5779)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5920..5942
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5936..6127)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(7439..8521)
                     /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     8483..8502
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(8522..8599)
                     /gene="lacI"
                     /label=lacI promoter
ORIGIN
        1 gaagtggcga gcccgatctt ccccatcggt gatgtcggcg atataggcgc cagcaaccgc
       61 acctgtggcg ccggtgatgc cggccacgat gcgtccggcg tagaggatcg agatctcgat
      121 cccgcgaaat taatacgact cactataggg gaattgtgag cggataacaa ttcccctcta
      181 gaaataattt tgtttaactt taagaaggag atataccatg ggcagcagcc atcatcatca
      241 tcatcacagc agcggcctgg tgccgcgcgg cagccatatg gctagctgga gccatccgca
      301 gtttgaaaaa ggtggtggta gcggtggtgg ttcaggtggt agtgcatggt cacaccctca
      361 gtttgagaaa atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga
      421 agtcaagcct gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt
      481 caagatcaaa aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg
      541 taaggaaatg gactccttaa gattcttgta cgacggtatt agaatccaag ctgatcagac
      601 ccctgaagat ttggacatgg aggataacga tattattgag gctcacagag aacagattgg
      661 tggatccaaa gagcaatttg aacaatgggt tgctcgcctt caaatatccg aggcagggca
      721 aaaaattatt gagaaagttc gctcatctga gccatcccgt cgcgttggtg gcggtagcaa
      781 gaatgtttcg ggacgctacc ctagtcgcaa gatgggagtc acgattcaat ttgagtctca
      841 tcgggttgaa ctgccaatta tctatcagtt agaacatgat aaagatgttc tagaattcta
      901 cgatcaacca ccgcaaatta aacttgacta tcaagcaagt aacggacgta gattaggcgt
      961 tttacacacc ccagatttat ttgtcatccg aaccaattcg gctggttggg aagagtgtaa
     1021 aactgagcag gatttgaaaa aactagcaga gaaaaacccc aatcgttact tttactctga
     1081 agaaaataat caatggcatt ccttgccagg agaagtttat gctaaccagt ttgggcttta
     1141 tcaccgcatt cattcagatc gcgaaatcaa ttgggttctg caacgtaatt tgcaattttt
     1201 agaagattat tatcgctcag aatctctagt agtggaagaa gcgatcgctc aatcattact
     1261 tgcaattgtg tcatctcaac caggaatcac tttagcagaa ttactcaatc ggtcaacagg
     1321 agctaaatct gacgatatct acaccttaat tattcaagag caaatctata ttgatttaaa
     1381 tgctacacca ctagctgaac cagaaagatg tctaatcttc cgtgacgagc aaactgctag
     1441 tgcttataga ttaatggttg agcaaccaag tgtaagtctt ccggctatct ctcctgtagt
     1501 caatattgtt actggagcaa ctgttagttg ggacggcaaa ggtttaaata tcatccatgt
     1561 tggagaaaca gaggttatac ttggcgcaga aaataatcag ttaattgaac tcaaaaaagc
     1621 tatatttgag aatctggttc gccaaggaaa aatcacaagt ctccaaacac cagaaaaaac
     1681 agctattagc acagaatctt ggcagcgatt tcatcaagct agtcctgaag accaagcaga
     1741 agcccttgaa cgctacaaaa ctatcgaacc ttacttaaac ggacatcctc cagaaaacga
     1801 aacaatacca gcccgtacca ttcgtcactg gaaagctaaa tatttaattg caattcaaaa
     1861 atacggttgc ggctacattg ggcttttgtc ccatcgcagt gtcaaaggta atcgacagcg
     1921 caagctgccc gaagacacct tagcaattat ggagaagttt attttagaag actatgaaac
     1981 tcttaaacaa aaaagaatgt gggaggttca cgcggcttta gttcgagctt gcgaagaagc
     2041 tggtgtaatt gctccgagct ataaagcatt taccaaagaa gtacaacgcc ggactggtta
     2101 cgaacagact aaaaaacgcc aaggtcgtcg tgccgcttat cagcatgaat ccttctattg
     2161 ggagcttgct ataactaccc caagacacgg cgatcgccct tttgaaattg ggcatataga
     2221 tcatactgag ttagatgtag agctagtttg ctctgataca ggtcgcaatc ttggcagacc
     2281 ttgggcaaca tttctcgtag atgcttattc acggcgattg ctggcagttt acctaacgtt
     2341 tgactcaccc tcatatcgtt cttgcttaat ggtgctaaga atttgtgtca agcgtcatgg
     2401 caggcttccc caaattgtag tggtagataa cggtgcagag tttcacagcg tttactttga
     2461 aacactacta gctacttttg aatgtacaaa aaagcaaaga cccccagcca aagctcgttt
     2521 tggttcggtt tgcgaacgcc tatttggaac ttccaatact caatttgtcc ataacttact
     2581 tggtaataca caaatcactc gtaatgtccg ccaagtcact aagtcagtta accctaaaaa
     2641 tctagctgta tggacacttg gtttacttta tgaatatctt tgtgcttggg cttacgaagt
     2701 ttatgataca gacgaacatc cggcactttt ccagagtccg agggatgcgt ttgctgcggg
     2761 tatggcgatt ggaggcagtc gggttcaccg catgattcct tacgatgaaa actttcagat
     2821 tcttacccta cctacaacct cagagggaaa agcaaaagtt caagttggac gcggcataaa
     2881 aattaactac atatactact ggtctaatag tttccgcgat cctcaaattg aaaatacctc
     2941 tgtgcagata agatacgacc cgtttaatat tggcatcgct tacgcctttg ttcgaggaca
     3001 gtgggtacag tgtatctctc aatattatgc agaactccaa ggtcgttcag aaaaagaact
     3061 gaagttagct agtatcgagt tacgcaaaca ctattcaaat catacacaac aatctaaagt
     3121 ttcggctaag aatttagcag aatttttagc atcggtagaa gctcaagaag ctctattaga
     3181 acagcgttcc tatgatgcag aagttaagga agtctttagg gtaatcgagg gtggaaaagc
     3241 gacaacaagc cgtaacgaag aaccaaaact tattcaagtt acttttgcca ataccgattt
     3301 tcaagcggat gaagatgaag cgatcgtccc agaaacactt gtagtgtatg aggagtttta
     3361 agcggccgca ctcgaggccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata
     3421 actagcataa ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg
     3481 aactatatcc ggatatcccg caagaggccc ggcagtaccg gcataaccaa gcctatgcct
     3541 acagcatcca gggtgacggt gccgaggatg acgatgagcg cattgttaga tttcatacac
     3601 ggtgcctgac tgcgttagca atttaactgt gataaactac cgcattaaag cttatcgatg
     3661 ataagctgtc aaacatgaga attcttgaag acgaaagggc ctcgtgatac gcctattttt
     3721 ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa
     3781 tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat
     3841 gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca
     3901 acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca
     3961 cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta
     4021 catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt
     4081 tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc
     4141 cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc
     4201 accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc
     4261 cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa
     4321 ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga
     4381 accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat
     4441 ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca
     4501 attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc
     4561 ggctggctgg tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat
     4621 tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag
     4681 tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa
     4741 gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca
     4801 tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc
     4861 ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc
     4921 ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc
     4981 agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt
     5041 cagcagagcg cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt
     5101 caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc
     5161 tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa
     5221 ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac
     5281 ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg
     5341 gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga
     5401 gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact
     5461 tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa
     5521 cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc
     5581 gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg
     5641 ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat
     5701 gcggtatttt ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt
     5761 acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact
     5821 gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc
     5881 tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga
     5941 ggttttcacc gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt
     6001 cgtgaagcga ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca
     6061 gaagcgttaa tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt
     6121 tggtcactga tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga
     6181 aacgagagag gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac
     6241 gttgtgaggg taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg
     6301 gtcaatgcca gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc
     6361 ctgcgatgca gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt
     6421 acgaaacacg gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc
     6481 agcagtcgct tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac
     6541 cccgccagcc tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg
     6601 acccaacgct gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata
     6661 tgttctgcca agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa
     6721 ttcttggagt ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc
     6781 ccggctccat gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac
     6841 aatccatgcc aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag
     6901 cggtccaatg atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg
     6961 atggtcgtca tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc
     7021 ggaagcgaga agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa
     7081 gacgtagccc agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg
     7141 tttggtggcg ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc
     7201 aagcgacagg ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca
     7261 gagcgctgcc ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc
     7321 gacgatagtc atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg
     7381 catcggtcga gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc
     7441 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     7501 cgcggggaga ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga
     7561 cgggcaacag ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca
     7621 cgctggtttg ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac
     7681 atgagctgtc ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc
     7741 cggactcggt aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg
     7801 cagtgggaac gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac
     7861 tccagtcgcc ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc
     7921 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
     7981 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
     8041 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
     8101 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
     8161 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
     8221 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
     8281 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
     8341 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
     8401 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
     8461 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
     8521 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
     8581 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
     8641 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
     8701 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
     8761 caagcgctca tgagccc
//
