LOCUS       sequence-330727-        7991 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AvTnsD expression construct for protein purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7991)
  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 7991)
  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: pMS17: pTwinStrep-SUMO-AvTnsD
FEATURES             Location/Qualifiers
     source          1..7991
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     568..587
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(607..684)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     890..909
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     993..1012
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        993..1011
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1012..1036
                     /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             1051..1073
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1092..1109
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1119..1136
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(1149..1172,1173..1208,1209..1232)
                     /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             1233..1526
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(3484..3502)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      3498..3545
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3720..3725,3726..3742,3743..3748))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3775..3793)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3861..3965
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3966..4034,4035..4826)
                     /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(4184..4203)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4997..5585
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5486..5505
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5739..5756
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5771..5910
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5836..5855)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5996..6018
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(6012..6203)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7515..7991,1..606))
                     /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 atgccagcca gccagacgca gacgcgccga gacagaactt aatgggcccg ctaacagcgc
       61 gatttgctgg tgacccaatg cgaccagatg ctccacgccc agtcgcgtac cgtcttcatg
      121 ggagaaaata atactgttga tgggtgtctg gtcagagaca tcaagaaata acgccggaac
      181 attagtgcag gcagcttcca cagcaatggc atcctggtca tccagcggat agttaatgat
      241 cagcccactg acgcgttgcg cgagaagatt gtgcaccgcc gctttacagg cttcgacgcc
      301 gcttcgttct accatcgaca ccaccacgct ggcacccagt tgatcggcgc gagatttaat
      361 cgccgcgaca atttgcgacg gcgcgtgcag ggccagactg gaggtggcaa cgccaatcag
      421 caacgactgt ttgcccgcca gttgttgtgc cacgcggttg ggaatgtaat tcagctccgc
      481 catcgccgct tccacttttt cccgcgtttt cgcagaaacg tggctggcct ggttcaccac
      541 gcgggaaacg gtctgataag agacaccggc atactctgcg acatcgtata acgttactgg
      601 tttcacattc accaccctga attgactctc ttccgggcgc tatcatgcca taccgcgaaa
      661 ggttttgcgc cattcgatgg tgtccgggat ctcgacgctc tcccttatgc gactcctgca
      721 ttaggaagca gcccagtagt aggttgaggc cgttgagcac cgccgccgca aggaatggtg
      781 catgcaagga gatggcgccc aacagtcccc cggccacggg gcctgccacc atacccacgc
      841 cgaaacaagc gctcatgagc ccgaagtggc gagcccgatc ttccccatcg gtgatgtcgg
      901 cgatataggc gccagcaacc gcacctgtgg cgccggtgat gccggccacg atgcgtccgg
      961 cgtagaggat cgagatctcg atcccgcgaa attaatacga ctcactatag gggaattgtg
     1021 agcggataac aattcccctc tagaaataat tttgtttaac tttaagaagg agatatacca
     1081 tgggcagcag ccatcatcat catcatcaca gcagcggcct ggtgccgcgc ggcagccata
     1141 tggctagctg gagccatccg cagtttgaaa aaggtggtgg tagcggtggt ggttcaggtg
     1201 gtagtgcatg gtcacaccct cagtttgaga aaatgtcgga ctcagaagtc aatcaagaag
     1261 ctaagccaga ggtcaagcca gaagtcaagc ctgagactca catcaattta aaggtgtccg
     1321 atggatcttc agagatcttc ttcaagatca aaaagaccac tcctttaaga aggctgatgg
     1381 aagcgttcgc taaaagacag ggtaaggaaa tggactcctt aagattcttg tacgacggta
     1441 ttagaatcca agctgatcag acccctgaag atttggacat ggaggataac gatattattg
     1501 aggctcacag agaacagatt ggtggatccg tgagtttttt ccctcaacct tatccagatg
     1561 aaattttata tagtgttatt gctcgttatc acattagaag tggcaatacg agtccaaaaa
     1621 ttacattaca agatttattt aagtcacaga ctactgtggc gacagtagat ttaccatcga
     1681 accttagtaa tttaattaaa aacctacaat ttatctcaaa ttaccaagtt gaagagttga
     1741 tatataaaca tactctctac cctttatata gccctttttt gccagcaaac aggtctagtc
     1801 aggtactgga atctatgaaa ggagattatg ggggagatat tcatacaaga gccggaatta
     1861 tggcaagctc aattactatg ccgaaatatt tgcggttttg tccaacttgc tgggaagaag
     1921 acttaaaaaa ttatggcgaa ccttactggc atcgcatcca tcaaatacca gggattttag
     1981 tttgtccatt tcatggtgaa gttttgcaag atagcctagc tcctttacat ggttttaata
     2041 agcatgaata ttatgcagct agtacagaca actgttctat tactaaagaa cccagtattc
     2101 ttagccctga cacactgaaa aaattactaa tattagccaa agatttttgg tggttactca
     2161 atagtgaaat accttgccaa gagccacagt ggtttcgcaa acagtacaca aatctgatga
     2221 ttgagcaggg ttttgcaact ccaactggtc gaatttacca gaacaaacta ctagataatt
     2281 ttttgttctt ttatggtcaa gaaatgctca gtgctgttga ttcaatggta aatcataatg
     2341 attcaaacaa ctgggttact agcatcgtca ggaaatatag gaaatctttc catcctctta
     2401 gacatctgct aatgatgcgt tttttgaaaa catcagtaga ggaatttttt caaaaagata
     2461 ctgaatacaa gccttttggc gaacccccct ggttgtgtct caatgctgcc gcagaacatt
     2521 atctacaacc agtgattacc aatttagtca ttactttatg ctgcgataca aaaaagcctg
     2581 tcggtacttt ttcttgtgct tgcggaatgg tttattgccg gactgggcct gatgaaacgg
     2641 atgaagacaa agttcgcatt ggtaaagtca aggcattcgg tcaaatatgg gagcaaaagc
     2701 tgaaagaatt ggtggaagtt gaaaagttgg gtttgcggga gacagccaga agattaaatg
     2761 ttgatgccag aactatcaaa cgttatgtat ctcgtctaaa attgacaact tattggcaga
     2821 ctagaacaga aaatgactct gtaggcttac agggagtccc agaaatagat cccaattttg
     2881 caactgaact caaattaaaa cacaggcaaa attggatggc tttacaagta cagaaccctg
     2941 aagcttcaaa gacaactctg agaaaactag taccagccac ctatatttgg ctatacagga
     3001 atgacagaga atggctcaat caaaactcac ccactttaca agttgccgtt ccttccgttg
     3061 tgagagttga ttggcatgag cgagatcagc aaattctcgc tcaggttcaa gatgcagtgc
     3121 gatcgctact taaggctgat ataccagaga aaatttctat tagtagggta gggaaaacta
     3181 tcggcttact tgcattactc gaaaaacatc tcgaccagat gcctttaaca aaggcttatt
     3241 tggaatcagt cgttgaaact gtagaagaat ttcagatgcg ccggatcaag tgggcaatca
     3301 agctgcttga tgaatgtgga gaagaaatta tgcgctggaa ggtagtcaga gttgctgctt
     3361 tgcgagagga ttgctcccaa agagtcagtg catttctgga aagtgaactg gagaaagcat
     3421 acaagaaaga gaggtgagcg gccgcactcg aggcccgaaa ggaagctgag ttggctgctg
     3481 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     3541 ttttgctgaa aggaggaact atatccggat atcccgcaag aggcccggca gtaccggcat
     3601 aaccaagcct atgcctacag catccagggt gacggtgccg aggatgacga tgagcgcatt
     3661 gttagatttc atacacggtg cctgactgcg ttagcaattt aactgtgata aactaccgca
     3721 ttaaagctta tcgatgataa gctgtcaaac atgagaattc ttgaagacga aagggcctcg
     3781 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     3841 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     3901 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     3961 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     4021 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     4081 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     4141 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     4201 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     4261 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     4321 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     4381 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     4441 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     4501 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     4561 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     4621 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     4681 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     4741 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     4801 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     4861 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     4921 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     4981 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     5041 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     5101 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     5161 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     5221 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     5281 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     5341 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     5401 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     5461 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     5521 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     5581 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     5641 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     5701 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     5761 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     5821 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagt atacactccg
     5881 ctatcgctac gtgactgggt catggctgcg ccccgacacc cgccaacacc cgctgacgcg
     5941 ccctgacggg cttgtctgct cccggcatcc gcttacagac aagctgtgac cgtctccggg
     6001 agctgcatgt gtcagaggtt ttcaccgtca tcaccgaaac gcgcgaggca gctgcggtaa
     6061 agctcatcag cgtggtcgtg aagcgattca cagatgtctg cctgttcatc cgcgtccagc
     6121 tcgttgagtt tctccagaag cgttaatgtc tggcttctga taaagcgggc catgttaagg
     6181 gcggtttttt cctgtttggt cactgatgcc tccgtgtaag ggggatttct gttcatgggg
     6241 gtaatgatac cgatgaaacg agagaggatg ctcacgatac gggttactga tgatgaacat
     6301 gcccggttac tggaacgttg tgagggtaaa caactggcgg tatggatgcg gcgggaccag
     6361 agaaaaatca ctcagggtca atgccagcgc ttcgttaata cagatgtagg tgttccacag
     6421 ggtagccagc agcatcctgc gatgcagatc cggaacataa tggtgcaggg cgctgacttc
     6481 cgcgtttcca gactttacga aacacggaaa ccgaagacca ttcatgttgt tgctcaggtc
     6541 gcagacgttt tgcagcagca gtcgcttcac gttcgctcgc gtatcggtga ttcattctgc
     6601 taaccagtaa ggcaaccccg ccagcctagc cgggtcctca acgacaggag cacgatcatg
     6661 cgcacccgtg gccaggaccc aacgctgccc gagatgcgcc gcgtgcggct gctggagatg
     6721 gcggacgcga tggatatgtt ctgccaaggg ttggtttgcg cattcacagt tctccgcaag
     6781 aattgattgg ctccaattct tggagtggtg aatccgttag cgaggtgccg ccggcttcca
     6841 ttcaggtcga ggtggcccgg ctccatgcac cgcgacgcaa cgcggggagg cagacaaggt
     6901 atagggcggc gcctacaatc catgccaacc cgttccatgt gctcgccgag gcggcataaa
     6961 tcgccgtgac gatcagcggt ccaatgatcg aagttaggct ggtaagagcc gcgagcgatc
     7021 cttgaagctg tccctgatgg tcgtcatcta cctgcctgga cagcatggcc tgcaacgcgg
     7081 gcatcccgat gccgccggaa gcgagaagaa tcataatggg gaaggccatc cagcctcgcg
     7141 tcgcgaacgc cagcaagacg tagcccagcg cgtcggccgc catgccggcg ataatggcct
     7201 gcttctcgcc gaaacgtttg gtggcgggac cagtgacgaa ggcttgagcg agggcgtgca
     7261 agattccgaa taccgcaagc gacaggccga tcatcgtcgc gctccagcga aagcggtcct
     7321 cgccgaaaat gacccagagc gctgccggca cctgtcctac gagttgcatg ataaagaaga
     7381 cagtcataag tgcggcgacg atagtcatgc cccgcgccca ccggaaggag ctgactgggt
     7441 tgaaggctct caagggcatc ggtcgagatc ccggtgccta atgagtgagc taacttacat
     7501 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt
     7561 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgccag ggtggttttt
     7621 cttttcacca gtgagacggg caacagctga ttgcccttca ccgcctggcc ctgagagagt
     7681 tgcagcaagc ggtccacgct ggtttgcccc agcaggcgaa aatcctgttt gatggtggtt
     7741 aacggcggga tataacatga gctgtcttcg gtatcgtcgt atcccactac cgagatatcc
     7801 gcaccaacgc gcagcccgga ctcggtaatg gcgcgcattg cgcccagcgc catctgatcg
     7861 ttggcaacca gcatcgcagt gggaacgatg ccctcattca gcatttgcat ggtttgttga
     7921 aaaccggaca tggcactcca gtcgccttcc cgttccgcta tcggctgaat ttgattgcga
     7981 gtgagatatt t
//
