LOCUS       sequence-330380-        7232 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  PmcTnsC expression construct for protein purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7232)
  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 7232)
  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: pMS32: pTwinStrep-SUMO-PmcTnsC
FEATURES             Location/Qualifiers
     source          1..7232
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    92..231
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     147..166
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(246..263)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(417..1005)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(497..516)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(1176..1967,1968..2036))
                     /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     1799..1818
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(2037..2141)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     2209..2227
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        join(2254..2259,2260..2276,2277..2282)
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     terminator      complement(2457..2504)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     2500..2518
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(3717..4010)
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     CDS             complement(join(4011..4034,4035..4070,4071..4094))
                     /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             complement(4107..4124)
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             complement(4134..4151)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     RBS             4170..4192
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    4207..4231
                     /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)."
     primer_bind     complement(4231..4250)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(4232..4250)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(4334..4353)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        4559..4636
                     /gene="lacI"
                     /label=lacI promoter
     CDS             4637..5719
                     /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     complement(4656..4675)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             7031..7222
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(join(7216..7232,1..6))
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
ORIGIN
        1 tcccggagac ggtcacagct tgtctgtaag cggatgccgg gagcagacaa gcccgtcagg
       61 gcgcgtcagc gggtgttggc gggtgtcggg gcgcagccat gacccagtca cgtagcgata
      121 gcggagtgta tactggctta actatgcggc atcagagcag attgtactga gagtgcacca
      181 ttgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc
      241 gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct
      301 cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg
      361 tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc
      421 cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga
      481 aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct
      541 cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg
      601 gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag
      661 ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat
      721 cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac
      781 aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac
      841 tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc
      901 ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt
      961 tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc
     1021 ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg
     1081 agattatcaa aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca
     1141 atctaaagta tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca
     1201 cctatctcag cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag
     1261 ataactacga tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac
     1321 ccacgctcac cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc
     1381 agaagtggtc ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct
     1441 agagtaagta gttcgccagt taatagtttg cgcaacgttg ttgccattgc tgcaggcatc
     1501 gtggtgtcac gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg
     1561 cgagttacat gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc
     1621 gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat
     1681 tctcttactg tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag
     1741 tcattctgag aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aacacgggat
     1801 aataccgcgc cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg
     1861 cgaaaactct caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca
     1921 cccaactgat cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga
     1981 aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc
     2041 ttcctttttc aatattattg aagcatttat cagggttatt gtctcatgag cggatacata
     2101 tttgaatgta tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg
     2161 ccacctgacg tctaagaaac cattattatc atgacattaa cctataaaaa taggcgtatc
     2221 acgaggccct ttcgtcttca agaattctca tgtttgacag cttatcatcg ataagcttta
     2281 atgcggtagt ttatcacagt taaattgcta acgcagtcag gcaccgtgta tgaaatctaa
     2341 caatgcgctc atcgtcatcc tcggcaccgt caccctggat gctgtaggca taggcttggt
     2401 tatgccggta ctgccgggcc tcttgcggga tatccggata tagttcctcc tttcagcaaa
     2461 aaacccctca agacccgttt agaggcccca aggggttatg ctagttattg ctcagcggtg
     2521 gcagcagcca actcagcttc ctttcgggcc tcgagtgcgg ccgcctagct aacatcttgc
     2581 tgcactccga tgggatctct tttaggtttg cgtttgccaa cctttcctgg cggacgagta
     2641 gtttttgggg tttcttcggg tagcacaatt ggttttgcgc caagtcctaa tgcagagcgt
     2701 aagttttgca catctgcttc tgtttcagat aattgtcgtt caccctcttg aatttccttg
     2761 aacattttct gacactgagc aaccgacaaa gcacgttttt gtaaatcttt gagagtgatt
     2821 gttgttgctt ctcgatccaa agcatcacta agaacacgct tcagccaatc tttcagtgtg
     2881 ccaatacatc ctaaagtacg ttcgtaaaaa tattcccaat gatcgactaa atttggagtt
     2941 tctgctagtg gcagatgctg ttgaaaagtt aacaacacac tcttaaaagc ctgtacatct
     3001 tctgggctat cagcacaata acggcggaaa tgaatatcaa cactacgacg tgagagttga
     3061 ccgctcaaat tacggaatgt aagtagttcg tatgttccta atagacagtg caagatgcct
     3121 gtcatatttg ctaaagattt gagacagtct aattgatctt gtagtttgta tccgcttgcc
     3181 accttaccaa aatgttgtgc ttcatctaca aaaaatacgt ctggatgacg atggatgaga
     3241 gcattttcta aagctctgcg tagagcaggt gcaactactt tcgactcaac attaattttg
     3301 ccaaaattat cgcgagagat accacgcaca ccgtaatcaa acttgtgatc gattaatggt
     3361 tcttccaaag taattaaggc gcgtgtgtag tattctttcc agttgaagta tctcgattct
     3421 ggcgctatag cttcaatacc aacaacagga acacgagcgc gatcgctttc caatttaggt
     3481 aatgctagtt ctgttaattt ctgctcaacg cggagtcgca aagttgtttt accaacacca
     3541 ctagcaccgt aaacaaagat aaatgaagct cctgctggtt cggcaatcgt tcgcatgaga
     3601 atttcataga cttctttaag tcgaggatgc gctacggtgt aattttcaaa gtaagcaagc
     3661 ctttctgtag caggccgtgt gaggagttca agcggaaatc cagtagattt tgtggatcca
     3721 ccaatctgtt ctctgtgagc ctcaataata tcgttatcct ccatgtccaa atcttcaggg
     3781 gtctgatcag cttggattct aataccgtcg tacaagaatc ttaaggagtc catttcctta
     3841 ccctgtcttt tagcgaacgc ttccatcagc cttcttaaag gagtggtctt tttgatcttg
     3901 aagaagatct ctgaagatcc atcggacacc tttaaattga tgtgagtctc aggcttgact
     3961 tctggcttga cctctggctt agcttcttga ttgacttctg agtccgacat tttctcaaac
     4021 tgagggtgtg accatgcact accacctgaa ccaccaccgc taccaccacc tttttcaaac
     4081 tgcggatggc tccagctagc catatggctg ccgcgcggca ccaggccgct gctgtgatga
     4141 tgatgatgat ggctgctgcc catggtatat ctccttctta aagttaaaca aaattatttc
     4201 tagaggggaa ttgttatccg ctcacaattc ccctatagtg agtcgtatta atttcgcggg
     4261 atcgagatct cgatcctcta cgccggacgc atcgtggccg gcatcaccgg cgccacaggt
     4321 gcggttgctg gcgcctatat cgccgacatc accgatgggg aagatcgggc tcgccacttc
     4381 gggctcatga gcgcttgttt cggcgtgggt atggtggcag gccccgtggc cgggggactg
     4441 ttgggcgcca tctccttgca tgcaccattc cttgcggcgg cggtgctcaa cggcctcaac
     4501 ctactactgg gctgcttcct aatgcaggag tcgcataagg gagagcgtcg agatcccgga
     4561 caccatcgaa tggcgcaaaa cctttcgcgg tatggcatga tagcgcccgg aagagagtca
     4621 attcagggtg gtgaatgtga aaccagtaac gttatacgat gtcgcagagt atgccggtgt
     4681 ctcttatcag accgtttccc gcgtggtgaa ccaggccagc cacgtttctg cgaaaacgcg
     4741 ggaaaaagtg gaagcggcga tggcggagct gaattacatt cccaaccgcg tggcacaaca
     4801 actggcgggc aaacagtcgt tgctgattgg cgttgccacc tccagtctgg ccctgcacgc
     4861 gccgtcgcaa attgtcgcgg cgattaaatc tcgcgccgat caactgggtg ccagcgtggt
     4921 ggtgtcgatg gtagaacgaa gcggcgtcga agcctgtaaa gcggcggtgc acaatcttct
     4981 cgcgcaacgc gtcagtgggc tgatcattaa ctatccgctg gatgaccagg atgccattgc
     5041 tgtggaagct gcctgcacta atgttccggc gttatttctt gatgtctctg accagacacc
     5101 catcaacagt attattttct cccatgaaga cggtacgcga ctgggcgtgg agcatctggt
     5161 cgcattgggt caccagcaaa tcgcgctgtt agcgggccca ttaagttctg tctcggcgcg
     5221 tctgcgtctg gctggctggc ataaatatct cactcgcaat caaattcagc cgatagcgga
     5281 acgggaaggc gactggagtg ccatgtccgg ttttcaacaa accatgcaaa tgctgaatga
     5341 gggcatcgtt cccactgcga tgctggttgc caacgatcag atggcgctgg gcgcaatgcg
     5401 cgccattacc gagtccgggc tgcgcgttgg tgcggatatc tcggtagtgg gatacgacga
     5461 taccgaagac agctcatgtt atatcccgcc gttaaccacc atcaaacagg attttcgcct
     5521 gctggggcaa accagcgtgg accgcttgct gcaactctct cagggccagg cggtgaaggg
     5581 caatcagctg ttgcccgtct cactggtgaa aagaaaaacc accctggcgc ccaatacgca
     5641 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     5701 actggaaagc gggcagtgag cgcaacgcaa ttaatgtaag ttagctcact cattaggcac
     5761 cgggatctcg accgatgccc ttgagagcct tcaacccagt cagctccttc cggtgggcgc
     5821 ggggcatgac tatcgtcgcc gcacttatga ctgtcttctt tatcatgcaa ctcgtaggac
     5881 aggtgccggc agcgctctgg gtcattttcg gcgaggaccg ctttcgctgg agcgcgacga
     5941 tgatcggcct gtcgcttgcg gtattcggaa tcttgcacgc cctcgctcaa gccttcgtca
     6001 ctggtcccgc caccaaacgt ttcggcgaga agcaggccat tatcgccggc atggcggccg
     6061 acgcgctggg ctacgtcttg ctggcgttcg cgacgcgagg ctggatggcc ttccccatta
     6121 tgattcttct cgcttccggc ggcatcggga tgcccgcgtt gcaggccatg ctgtccaggc
     6181 aggtagatga cgaccatcag ggacagcttc aaggatcgct cgcggctctt accagcctaa
     6241 cttcgatcat tggaccgctg atcgtcacgg cgatttatgc cgcctcggcg agcacatgga
     6301 acgggttggc atggattgta ggcgccgccc tataccttgt ctgcctcccc gcgttgcgtc
     6361 gcggtgcatg gagccgggcc acctcgacct gaatggaagc cggcggcacc tcgctaacgg
     6421 attcaccact ccaagaattg gagccaatca attcttgcgg agaactgtga atgcgcaaac
     6481 caacccttgg cagaacatat ccatcgcgtc cgccatctcc agcagccgca cgcggcgcat
     6541 ctcgggcagc gttgggtcct ggccacgggt gcgcatgatc gtgctcctgt cgttgaggac
     6601 ccggctaggc tggcggggtt gccttactgg ttagcagaat gaatcaccga tacgcgagcg
     6661 aacgtgaagc gactgctgct gcaaaacgtc tgcgacctga gcaacaacat gaatggtctt
     6721 cggtttccgt gtttcgtaaa gtctggaaac gcggaagtca gcgccctgca ccattatgtt
     6781 ccggatctgc atcgcaggat gctgctggct accctgtgga acacctacat ctgtattaac
     6841 gaagcgctgg cattgaccct gagtgatttt tctctggtcc cgccgcatcc ataccgccag
     6901 ttgtttaccc tcacaacgtt ccagtaaccg ggcatgttca tcatcagtaa cccgtatcgt
     6961 gagcatcctc tctcgtttca tcggtatcat tacccccatg aacagaaatc ccccttacac
     7021 ggaggcatca gtgaccaaac aggaaaaaac cgcccttaac atggcccgct ttatcagaag
     7081 ccagacatta acgcttctgg agaaactcaa cgagctggac gcggatgaac aggcagacat
     7141 ctgtgaatcg cttcacgacc acgctgatga gctttaccgc agctgcctcg cgcgtttcgg
     7201 tgatgacggt gaaaacctct gacacatgca gc
//
