LOCUS       sequence-330383-        6899 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  AvTnsA expression construct for protein purification..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6899)
  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 6899)
  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: pMS14: pTwinStrep-SUMO-AvTnsA
FEATURES             Location/Qualifiers
     source          1..6899
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     563..582
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(602..679)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     885..904
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     988..1007
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        988..1006
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1007..1031
                     /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             1046..1068
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1087..1104
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             1114..1131
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(1144..1167,1168..1203,1204..1227)
                     /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             1228..1521
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(2387..2405)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2401..2448
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2623..2628,2629..2645,2646..2651))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2678..2696)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2764..2868
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2869..2937,2938..3729)
                     /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(3087..3106)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3900..4488
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4389..4408
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4642..4659
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4674..4813
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4739..4758)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4899..4921
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4915..5106)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6418..6899,1..601))
                     /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 agccagccag acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt
       61 gctggtgacc caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga
      121 aaataatact gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag
      181 tgcaggcagc ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc
      241 cactgacgcg ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc
      301 gttctaccat cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg
      361 cgacaatttg cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg
      421 actgtttgcc cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg
      481 ccgcttccac tttttcccgc gttttcgcag aaacgtggct ggcctggttc accacgcggg
      541 aaacggtctg ataagagaca ccggcatact ctgcgacatc gtataacgtt actggtttca
      601 cattcaccac cctgaattga ctctcttccg ggcgctatca tgccataccg cgaaaggttt
      661 tgcgccattc gatggtgtcc gggatctcga cgctctccct tatgcgactc ctgcattagg
      721 aagcagccca gtagtaggtt gaggccgttg agcaccgccg ccgcaaggaa tggtgcatgc
      781 aaggagatgg cgcccaacag tcccccggcc acggggcctg ccaccatacc cacgccgaaa
      841 caagcgctca tgagcccgaa gtggcgagcc cgatcttccc catcggtgat gtcggcgata
      901 taggcgccag caaccgcacc tgtggcgccg gtgatgccgg ccacgatgcg tccggcgtag
      961 aggatcgaga tctcgatccc gcgaaattaa tacgactcac tataggggaa ttgtgagcgg
     1021 ataacaattc ccctctagaa ataattttgt ttaactttaa gaaggagata taccatgggc
     1081 agcagccatc atcatcatca tcacagcagc ggcctggtgc cgcgcggcag ccatatggct
     1141 agctggagcc atccgcagtt tgaaaaaggt ggtggtagcg gtggtggttc aggtggtagt
     1201 gcatggtcac accctcagtt tgagaaaatg tcggactcag aagtcaatca agaagctaag
     1261 ccagaggtca agccagaagt caagcctgag actcacatca atttaaaggt gtccgatgga
     1321 tcttcagaga tcttcttcaa gatcaaaaag accactcctt taagaaggct gatggaagcg
     1381 ttcgctaaaa gacagggtaa ggaaatggac tccttaagat tcttgtacga cggtattaga
     1441 atccaagctg atcagacccc tgaagatttg gacatggagg ataacgatat tattgaggct
     1501 cacagagaac agattggtgg atccgttagg ggtagaaggg aatggactca agctaaatat
     1561 gagcgatata taaaagaagg tcgtggccga ggaagtggta agcactataa accttggctt
     1621 acaatccaag atgttccctc taaaggacgt tcttctagaa ctcctggttg gaaaacaaat
     1681 cgagtacatc atttcttctc agaccatgaa aaacggctat tttatttgtt tgagtggtca
     1741 gatatagtca cagatataag agagcaattt cctttagaca atctcgatct agccatgagt
     1801 attgctacag acataggtat aaagtaccct gtagatacac aaagtggcac tcctcacgtt
     1861 ttaacaagcg actttatgct tacagtcaac cgcgatggga agcagatgca gattgcccgg
     1921 actgttaagc cgtcaacaga actggagaaa aagcaagtta atgagaaatt cgagatagaa
     1981 agacaatatt acttagcaaa gggtattgac tggggaatta tcacagaaaa agaaattccc
     2041 aggcttttag ctgaaaatgt tgagtgggtt cattctgctt ataggctaga agcaaattct
     2101 gaaatggaca ctgcacaact gagtgaattg tcaacaattt taaaatctaa gcttcaggaa
     2161 gaagatacaa ctataaatag agtaactact gctttagaca aagaaatgaa tataaattcc
     2221 ggtacatcac tttatgtatt taaacattta attgctagga aagaaatcct tgtggatcta
     2281 gtagcaataa aattatctgc ttgtccgtca actaaagtta ttgaaaaaat aatattttag
     2341 gcggccgcac tcgaggcccg aaaggaagct gagttggctg ctgccaccgc tgagcaataa
     2401 ctagcataac cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga
     2461 actatatccg gatatcccgc aagaggcccg gcagtaccgg cataaccaag cctatgccta
     2521 cagcatccag ggtgacggtg ccgaggatga cgatgagcgc attgttagat ttcatacacg
     2581 gtgcctgact gcgttagcaa tttaactgtg ataaactacc gcattaaagc ttatcgatga
     2641 taagctgtca aacatgagaa ttcttgaaga cgaaagggcc tcgtgatacg cctattttta
     2701 taggttaatg tcatgataat aatggtttct tagacgtcag gtggcacttt tcggggaaat
     2761 gtgcgcggaa cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg
     2821 agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat gagtattcaa
     2881 catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt ttttgctcac
     2941 ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg agtgggttac
     3001 atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga agaacgtttt
     3061 ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg tgttgacgcc
     3121 gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt tgagtactca
     3181 ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg cagtgctgcc
     3241 ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg aggaccgaag
     3301 gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga tcgttgggaa
     3361 ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc tgcagcaatg
     3421 gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc ccggcaacaa
     3481 ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc ggcccttccg
     3541 gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg cggtatcatt
     3601 gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac gacggggagt
     3661 caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc actgattaag
     3721 cattggtaac tgtcagacca agtttactca tatatacttt agattgattt aaaacttcat
     3781 ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac caaaatccct
     3841 taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa aggatcttct
     3901 tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca
     3961 gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt aactggcttc
     4021 agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg ccaccacttc
     4081 aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc agtggctgct
     4141 gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt accggataag
     4201 gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga gcgaacgacc
     4261 tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg
     4321 agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg cacgagggag
     4381 cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca cctctgactt
     4441 gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac
     4501 gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt ctttcctgcg
     4561 ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga taccgctcgc
     4621 cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga gcgcctgatg
     4681 cggtattttc tccttacgca tctgtgcggt atttcacacc gcaatggtgc actctcagta
     4741 caatctgctc tgatgccgca tagttaagcc agtatacact ccgctatcgc tacgtgactg
     4801 ggtcatggct gcgccccgac acccgccaac acccgctgac gcgccctgac gggcttgtct
     4861 gctcccggca tccgcttaca gacaagctgt gaccgtctcc gggagctgca tgtgtcagag
     4921 gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg taaagctcat cagcgtggtc
     4981 gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc agctcgttga gtttctccag
     5041 aagcgttaat gtctggcttc tgataaagcg ggccatgtta agggcggttt tttcctgttt
     5101 ggtcactgat gcctccgtgt aagggggatt tctgttcatg ggggtaatga taccgatgaa
     5161 acgagagagg atgctcacga tacgggttac tgatgatgaa catgcccggt tactggaacg
     5221 ttgtgagggt aaacaactgg cggtatggat gcggcgggac cagagaaaaa tcactcaggg
     5281 tcaatgccag cgcttcgtta atacagatgt aggtgttcca cagggtagcc agcagcatcc
     5341 tgcgatgcag atccggaaca taatggtgca gggcgctgac ttccgcgttt ccagacttta
     5401 cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag gtcgcagacg ttttgcagca
     5461 gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc tgctaaccag taaggcaacc
     5521 ccgccagcct agccgggtcc tcaacgacag gagcacgatc atgcgcaccc gtggccagga
     5581 cccaacgctg cccgagatgc gccgcgtgcg gctgctggag atggcggacg cgatggatat
     5641 gttctgccaa gggttggttt gcgcattcac agttctccgc aagaattgat tggctccaat
     5701 tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt ccattcaggt cgaggtggcc
     5761 cggctccatg caccgcgacg caacgcgggg aggcagacaa ggtatagggc ggcgcctaca
     5821 atccatgcca acccgttcca tgtgctcgcc gaggcggcat aaatcgccgt gacgatcagc
     5881 ggtccaatga tcgaagttag gctggtaaga gccgcgagcg atccttgaag ctgtccctga
     5941 tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg cgggcatccc gatgccgccg
     6001 gaagcgagaa gaatcataat ggggaaggcc atccagcctc gcgtcgcgaa cgccagcaag
     6061 acgtagccca gcgcgtcggc cgccatgccg gcgataatgg cctgcttctc gccgaaacgt
     6121 ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt gcaagattcc gaataccgca
     6181 agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt cctcgccgaa aatgacccag
     6241 agcgctgccg gcacctgtcc tacgagttgc atgataaaga agacagtcat aagtgcggcg
     6301 acgatagtca tgccccgcgc ccaccggaag gagctgactg ggttgaaggc tctcaagggc
     6361 atcggtcgag atcccggtgc ctaatgagtg agctaactta cattaattgc gttgcgctca
     6421 ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc
     6481 gcggggagag gcggtttgcg tattgggcgc cagggtggtt tttcttttca ccagtgagac
     6541 gggcaacagc tgattgccct tcaccgcctg gccctgagag agttgcagca agcggtccac
     6601 gctggtttgc cccagcaggc gaaaatcctg tttgatggtg gttaacggcg ggatataaca
     6661 tgagctgtct tcggtatcgt cgtatcccac taccgagata tccgcaccaa cgcgcagccc
     6721 ggactcggta atggcgcgca ttgcgcccag cgccatctga tcgttggcaa ccagcatcgc
     6781 agtgggaacg atgccctcat tcagcatttg catggtttgt tgaaaaccgg acatggcact
     6841 ccagtcgcct tcccgttccg ctatcggctg aatttgattg cgagtgagat atttatgcc
//
