LOCUS       sequence-330859-        8309 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AvTnsB expression construct for protein purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8309)
  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 8309)
  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: pMS15: pTwinStrep-SUMO-AvTnsB
FEATURES             Location/Qualifiers
     source          1..8309
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     917..936
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(956..1033)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1239..1258
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1342..1361
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1342..1360
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1361..1385
                     /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             1400..1422
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1441..1458
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1468..1485
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(1498..1521,1522..1557,1558..1581)
                     /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             1582..1875
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(4151..4169)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      4165..4212
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(4387..4392,4393..4409,4410..4415))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(4442..4460)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4528..4632
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4633..4701,4702..5493)
                     /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(4851..4870)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5664..6252
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6153..6172
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     6406..6423
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    6438..6577
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(6503..6522)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6663..6685
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6679..6870)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(8182..8309,1..955))
                     /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 acagctgatt gcccttcacc gcctggccct gagagagttg cagcaagcgg tccacgctgg
       61 tttgccccag caggcgaaaa tcctgtttga tggtggttaa cggcgggata taacatgagc
      121 tgtcttcggt atcgtcgtat cccactaccg agatatccgc accaacgcgc agcccggact
      181 cggtaatggc gcgcattgcg cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg
      241 gaacgatgcc ctcattcagc atttgcatgg tttgttgaaa accggacatg gcactccagt
      301 cgccttcccg ttccgctatc ggctgaattt gattgcgagt gagatattta tgccagccag
      361 ccagacgcag acgcgccgag acagaactta atgggcccgc taacagcgcg atttgctggt
      421 gacccaatgc gaccagatgc tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa
      481 tactgttgat gggtgtctgg tcagagacat caagaaataa cgccggaaca ttagtgcagg
      541 cagcttccac agcaatggca tcctggtcat ccagcggata gttaatgatc agcccactga
      601 cgcgttgcgc gagaagattg tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta
      661 ccatcgacac caccacgctg gcacccagtt gatcggcgcg agatttaatc gccgcgacaa
      721 tttgcgacgg cgcgtgcagg gccagactgg aggtggcaac gccaatcagc aacgactgtt
      781 tgcccgccag ttgttgtgcc acgcggttgg gaatgtaatt cagctccgcc atcgccgctt
      841 ccactttttc ccgcgttttc gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg
      901 tctgataaga gacaccggca tactctgcga catcgtataa cgttactggt ttcacattca
      961 ccaccctgaa ttgactctct tccgggcgct atcatgccat accgcgaaag gttttgcgcc
     1021 attcgatggt gtccgggatc tcgacgctct cccttatgcg actcctgcat taggaagcag
     1081 cccagtagta ggttgaggcc gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag
     1141 atggcgccca acagtccccc ggccacgggg cctgccacca tacccacgcc gaaacaagcg
     1201 ctcatgagcc cgaagtggcg agcccgatct tccccatcgg tgatgtcggc gatataggcg
     1261 ccagcaaccg cacctgtggc gccggtgatg ccggccacga tgcgtccggc gtagaggatc
     1321 gagatctcga tcccgcgaaa ttaatacgac tcactatagg ggaattgtga gcggataaca
     1381 attcccctct agaaataatt ttgtttaact ttaagaagga gatataccat gggcagcagc
     1441 catcatcatc atcatcacag cagcggcctg gtgccgcgcg gcagccatat ggctagctgg
     1501 agccatccgc agtttgaaaa aggtggtggt agcggtggtg gttcaggtgg tagtgcatgg
     1561 tcacaccctc agtttgagaa aatgtcggac tcagaagtca atcaagaagc taagccagag
     1621 gtcaagccag aagtcaagcc tgagactcac atcaatttaa aggtgtccga tggatcttca
     1681 gagatcttct tcaagatcaa aaagaccact cctttaagaa ggctgatgga agcgttcgct
     1741 aaaagacagg gtaaggaaat ggactcctta agattcttgt acgacggtat tagaatccaa
     1801 gctgatcaga cccctgaaga tttggacatg gaggataacg atattattga ggctcacaga
     1861 gaacagattg gtggatccgt taaagattta tttgtcaatg atttaataga gtggattgta
     1921 taccaaaaca ctcatatagt cgaaagaatt atttggatag atgaaggtta cattattgca
     1981 tttgttatag atattaatgc taagaaaggt ctacctgaac caaggaaaat atcagatatt
     2041 ttagaagcta taactgaagg tattgctttt aagcagcaac aagatccttg ggcaagaatt
     2101 attcgggatg aaaatttgac agataaggaa aaggaataca gagataaagc ttgggagata
     2161 atttcatcat taattataga agaaccatca atatattata gaaattttag aggatcgatg
     2221 gttaaagaag cggttgaaaa gtacaacgaa gaaagggata aagataaatt agtagaaaaa
     2281 accgtctatg gttatttgag aagattttgg cagagaggaa aaactataaa tgctctcata
     2341 cctgatttta tcaattcatc aggtaagggc aagattcgag gacaatcggg caagaagaga
     2401 ggaagaccaa gaaaatatgc acatgaccca gatattggtg aaggtgtaaa tgttacagaa
     2461 gaagatagaa agatttttag agtatctata agcaaatttt acaataatcc aaaagaaaac
     2521 tacttaacaa cagcctatga attaatggtg aaagaatatt acaaggaagg aattagatat
     2581 gacgagaatg gtgttcaaaa atctatatta accccaactg ataaaatacc tacaattact
     2641 caattcaaat actggtatca acttgaacag gatgatgtta ccaaaacttt aacatctcga
     2701 aaaggagcca agcgagtagc tttagagaat agagcaattt tgggaaactc taagatggaa
     2761 actattggcc ctggttctag atatcaaatt gatgccacag ttgctgatgt atatttagtt
     2821 tcaaaatata accgtgactg gataattggt agaccagtta tctatgtaat tattgatgtt
     2881 tttagtagaa tgattacagg tgtttatgtc ggtttagaag gcccatcttg gataggagca
     2941 atgatggcac tagccaatgt agctacagat aaaaccctgt tttgccagga atataacatt
     3001 aatattactg aaaatgaatg gccttgcaat caaatgccag atgcaatttt aggagatagg
     3061 ggagaattag caggtatgac agtagaaaca ttaatcccta atctttatgt tcgtattgaa
     3121 aatgcagctt cctatagagc agattggaaa gggttagtag aaagacactt tcgtataatt
     3181 catgaatatg taaaaccttt tttacctggt tatgtggata cggattttag gcaaagaggt
     3241 gcaagggatt ataggcttga tggcaaactt gatatagatc aatttactga gattattatt
     3301 cgtttaattg ttcaccataa caataaacct cttactgaat atgaaagaga tgaagcaatg
     3361 attgctgatg atacccctcc tatacctaga gagttgtgga agtggggtat cgccaatcgt
     3421 tctggcagac tcagaacttt ttctgaagat attgtcaaat tgaacttgat gcctactgag
     3481 aaagcaacta ttactgctaa aggcatcaaa atgagaggta aggagatgta ctatacctgt
     3541 gatagtgcta aaaatgagca atggtttgaa aaagctagaa gtaatctcct ttctaagtca
     3601 gaaaaatcat tgattgtatc ttatgacatc agaaagccaa acttcattta tcttccatct
     3661 agtgatggta gagattttga aaaatgtttt cttattgact tagaagggcg gtatgctaat
     3721 aaaaactttc atgatattga atatctgctt gcttatgaag aattgcaaag acagaaaagc
     3781 caaggtaaac agctacaaga aaaggtagat ttaatagctg atattgaaag catagtagat
     3841 agagcagaaa aaatgactga tgcttctcaa tatcataatt taagtaagag caagaaagtt
     3901 tctggaataa gagataatag ggcttttgaa aaggctaaac gacgagagaa tgaaggattt
     3961 gaattagcaa ttacagataa tcagagcagt tttcaggttg cagagagtac agcagaaacc
     4021 tccgaacagc cagaatcact acagccaaac catctgaatc ttcttaagaa aaagcgacag
     4081 gagcgaaaac gtggacagaa ttgagcggcc gcactcgagg cccgaaagga agctgagttg
     4141 gctgctgcca ccgctgagca ataactagca taaccccttg gggcctctaa acgggtcttg
     4201 aggggttttt tgctgaaagg aggaactata tccggatatc ccgcaagagg cccggcagta
     4261 ccggcataac caagcctatg cctacagcat ccagggtgac ggtgccgagg atgacgatga
     4321 gcgcattgtt agatttcata cacggtgcct gactgcgtta gcaatttaac tgtgataaac
     4381 taccgcatta aagcttatcg atgataagct gtcaaacatg agaattcttg aagacgaaag
     4441 ggcctcgtga tacgcctatt tttataggtt aatgtcatga taataatggt ttcttagacg
     4501 tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata
     4561 cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga
     4621 aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca
     4681 ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat
     4741 cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag
     4801 agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc
     4861 gcggtattat cccgtgttga cgccgggcaa gagcaactcg gtcgccgcat acactattct
     4921 cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca
     4981 gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt
     5041 ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat
     5101 gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt
     5161 gacaccacga tgcctgcagc aatggcaaca acgttgcgca aactattaac tggcgaacta
     5221 cttactctag cttcccggca acaattaata gactggatgg aggcggataa agttgcagga
     5281 ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt
     5341 gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc
     5401 gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct
     5461 gagataggtg cctcactgat taagcattgg taactgtcag accaagttta ctcatatata
     5521 ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt
     5581 gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc
     5641 gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg
     5701 caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact
     5761 ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg
     5821 tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg
     5881 ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac
     5941 tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca
     6001 cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga
     6061 gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc
     6121 ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct
     6181 gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg
     6241 agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct
     6301 tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc
     6361 tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc
     6421 gaggaagcgg aagagcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca
     6481 caccgcaatg gtgcactctc agtacaatct gctctgatgc cgcatagtta agccagtata
     6541 cactccgcta tcgctacgtg actgggtcat ggctgcgccc cgacacccgc caacacccgc
     6601 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     6661 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgaggcagct
     6721 gcggtaaagc tcatcagcgt ggtcgtgaag cgattcacag atgtctgcct gttcatccgc
     6781 gtccagctcg ttgagtttct ccagaagcgt taatgtctgg cttctgataa agcgggccat
     6841 gttaagggcg gttttttcct gtttggtcac tgatgcctcc gtgtaagggg gatttctgtt
     6901 catgggggta atgataccga tgaaacgaga gaggatgctc acgatacggg ttactgatga
     6961 tgaacatgcc cggttactgg aacgttgtga gggtaaacaa ctggcggtat ggatgcggcg
     7021 ggaccagaga aaaatcactc agggtcaatg ccagcgcttc gttaatacag atgtaggtgt
     7081 tccacagggt agccagcagc atcctgcgat gcagatccgg aacataatgg tgcagggcgc
     7141 tgacttccgc gtttccagac tttacgaaac acggaaaccg aagaccattc atgttgttgc
     7201 tcaggtcgca gacgttttgc agcagcagtc gcttcacgtt cgctcgcgta tcggtgattc
     7261 attctgctaa ccagtaaggc aaccccgcca gcctagccgg gtcctcaacg acaggagcac
     7321 gatcatgcgc acccgtggcc aggacccaac gctgcccgag atgcgccgcg tgcggctgct
     7381 ggagatggcg gacgcgatgg atatgttctg ccaagggttg gtttgcgcat tcacagttct
     7441 ccgcaagaat tgattggctc caattcttgg agtggtgaat ccgttagcga ggtgccgccg
     7501 gcttccattc aggtcgaggt ggcccggctc catgcaccgc gacgcaacgc ggggaggcag
     7561 acaaggtata gggcggcgcc tacaatccat gccaacccgt tccatgtgct cgccgaggcg
     7621 gcataaatcg ccgtgacgat cagcggtcca atgatcgaag ttaggctggt aagagccgcg
     7681 agcgatcctt gaagctgtcc ctgatggtcg tcatctacct gcctggacag catggcctgc
     7741 aacgcgggca tcccgatgcc gccggaagcg agaagaatca taatggggaa ggccatccag
     7801 cctcgcgtcg cgaacgccag caagacgtag cccagcgcgt cggccgccat gccggcgata
     7861 atggcctgct tctcgccgaa acgtttggtg gcgggaccag tgacgaaggc ttgagcgagg
     7921 gcgtgcaaga ttccgaatac cgcaagcgac aggccgatca tcgtcgcgct ccagcgaaag
     7981 cggtcctcgc cgaaaatgac ccagagcgct gccggcacct gtcctacgag ttgcatgata
     8041 aagaagacag tcataagtgc ggcgacgata gtcatgcccc gcgcccaccg gaaggagctg
     8101 actgggttga aggctctcaa gggcatcggt cgagatcccg gtgcctaatg agtgagctaa
     8161 cttacattaa ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag
     8221 ctgcattaat gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgccagggt
     8281 ggtttttctt ttcaccagtg agacgggca
//
