LOCUS       sequence-266856-        8000 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Protein expression construct for ShCast12k.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8000)
  AUTHORS   Strecker J, Ladha A, Gardner Z, Schmid-Burgk JL, Makarova KS, Koonin
            EV, Zhang F
  TITLE     RNA-guided DNA insertion with CRISPR-associated transposases.
  JOURNAL   Science. 2019 Jul 5;365(6448):48-53. doi: 10.1126/science.aax9181. 
            Epub 2019 Jun 6.
  PUBMED    31171706
REFERENCE   2  (bases 1 to 8000)
  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: 
            "10.1126/science.aax9181"; journalName: "Science"; date: "2019-07-5-
            5"; volume: "365"; issue: "6448"; pages: "48-53"
COMMENT     Sequence Label: pXT128_TwinStrep-SUMO-ShCas12k
FEATURES             Location/Qualifiers
     source          1..8000
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    1..25
                     /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             40..62
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             81..98
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             108..125
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(138..161,162..197,198..221)
                     /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             222..515
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(2482..2500)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2496..2543
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2718..2723,2724..2740,2741..2746))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2773..2791)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2859..2963
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2964..3032,3033..3824)
                     /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(3182..3201)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3995..4583
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4484..4503
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4737..4754
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4769..4908
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4834..4853)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4994..5016
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5010..5201)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(6513..7595)
                     /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     7557..7576
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(7596..7673)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7879..7898
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     join(7982..8000,1)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        7982..8000
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 ggaattgtga gcggataaca attcccctct agaaataatt ttgtttaact ttaagaagga
       61 gatataccat gggcagcagc catcatcatc atcatcacag cagcggcctg gtgccgcgcg
      121 gcagccatat ggctagctgg agccatccgc agtttgaaaa aggtggtggt agcggtggtg
      181 gttcaggtgg tagtgcatgg tcacaccctc agtttgagaa aatgtcggac tcagaagtca
      241 atcaagaagc taagccagag gtcaagccag aagtcaagcc tgagactcac atcaatttaa
      301 aggtgtccga tggatcttca gagatcttct tcaagatcaa aaagaccact cctttaagaa
      361 ggctgatgga agcgttcgct aaaagacagg gtaaggaaat ggactcctta agattcttgt
      421 acgacggtat tagaatccaa gctgatcaga cccctgaaga tttggacatg gaggataacg
      481 atattattga ggctcacaga gaacagattg gtggatcaag ccagatcacc atccaggcca
      541 gactgatcag cttcgagagc aacagacagc agctgtggaa gctgatggcc gacctgaaca
      601 cccctctgat caacgagctg ctgtgtcagc tgggacagca ccccgatttc gagaagtggc
      661 agcagaaggg caagctgcct agcaccgtgg tgtctcagct gtgccagcct ctgaaaacag
      721 accccagatt tgccggccag cctagcagac tgtacatgag cgccatccac atcgtggact
      781 acatctacaa gagctggctg gccatccaga agcggctgca gcaacagctg gatggcaaga
      841 ccagatggct ggaaatgctg aacagcgacg ccgagctggt ggaactgtct ggcgatacac
      901 tggaagccat cagagtgaag gccgccgaga tcctggccat tgctatgcct gccagcgaga
      961 gcgatagcgc tagccccaaa ggcaaaaagg gcaagaaaga gaagaagccc tccagcagca
     1021 gccccaagag aagcctgagc aagaccctgt tcgacgccta ccaagagaca gaggacatca
     1081 agagcagaag cgccatctcc tacctgctga agaacggctg caagctgacc gacaaagaag
     1141 aggacagcga gaagttcgcc aagcggcgga gacaggtgga aatccagatc cagagactga
     1201 ccgagaagct gattagcaga atgcccaagg gccgcgacct gaccaatgcc aagtggctcg
     1261 aaacactgct gaccgccaca accacagtgg ccgaggataa tgcccaggcc aagagatggc
     1321 aggacatcct gctgaccaga agcagctccc tgccttttcc actggtgttc gagacaaacg
     1381 aggacatggt ctggtccaag aaccagaaag gccggctgtg cgtgcacttc aacggcctga
     1441 gcgatctgat cttcgaggtg tactgcggca accggcagct gcactggttc cagcggtttc
     1501 tggaagatca gcagaccaag cgcaagagca agaatcagca cagcagcggc ctgttcaccc
     1561 tgagaaatgg acacctcgtg tggctggaag gcgaaggcaa aggcgagcct tggaacctgc
     1621 accacctgac actgtactgc tgcgtggaca accggctgtg gacagaagag ggcacagaga
     1681 tcgtgcggca agagaaggcc gatgagatca ccaagttcat caccaacatg aagaagaagt
     1741 ccgacctgag cgacacccag caggccctga ttcagagaaa gcagagcacc ctgaccagga
     1801 tcaacaactc cttcgagagg cccagccagc cactgtatca gggccagtct catatcctcg
     1861 tgggcgtgtc actgggcctc gagaaacctg ctacagtcgc cgtggtggac gccattgcca
     1921 acaaggttct ggcctaccgg tccatcaaac agctgctcgg cgacaactat gagctgctga
     1981 accgtcagcg acggcagcag cagtatctga gccacgagag acacaaggcc cagaagaact
     2041 tcagccccaa ccagtttggc gcctctgaac tgggccagca catcgataga ctgctggcca
     2101 aggccattgt ggccctggcc agaacataca aggccggcag tatcgtgctg cccaagctgg
     2161 gcgatatgag agaagtggtg cagagcgaga tccaggctat cgccgaacag aagttccccg
     2221 gctatatcga gggccagcag aagtatgcca agcagtatag agtgaacgtg caccggtggt
     2281 cctacggcag gctgatccag agcatccagt ctaaggctgc ccagaccggc atcgtgatcg
     2341 aggaaggcaa gcagcccatt agaggcagcc ctcacgacaa ggctaaagaa ctggctctga
     2401 gcgcctacaa cctgaggctg acaagaagaa gctgagcggc cgcactcgag gcccgaaagg
     2461 aagctgagtt ggctgctgcc accgctgagc aataactagc ataacccctt ggggcctcta
     2521 aacgggtctt gaggggtttt ttgctgaaag gaggaactat atccggatat cccgcaagag
     2581 gcccggcagt accggcataa ccaagcctat gcctacagca tccagggtga cggtgccgag
     2641 gatgacgatg agcgcattgt tagatttcat acacggtgcc tgactgcgtt agcaatttaa
     2701 ctgtgataaa ctaccgcatt aaagcttatc gatgataagc tgtcaaacat gagaattctt
     2761 gaagacgaaa gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg
     2821 tttcttagac gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat
     2881 ttttctaaat acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc
     2941 aataatattg aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct
     3001 tttttgcggc attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag
     3061 atgctgaaga tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta
     3121 agatccttga gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc
     3181 tgctatgtgg cgcggtatta tcccgtgttg acgccgggca agagcaactc ggtcgccgca
     3241 tacactattc tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg
     3301 atggcatgac agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg
     3361 ccaacttact tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca
     3421 tgggggatca tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa
     3481 acgacgagcg tgacaccacg atgcctgcag caatggcaac aacgttgcgc aaactattaa
     3541 ctggcgaact acttactcta gcttcccggc aacaattaat agactggatg gaggcggata
     3601 aagttgcagg accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat
     3661 ctggagccgg tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc
     3721 cctcccgtat cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata
     3781 gacagatcgc tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt
     3841 actcatatat actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga
     3901 agatcctttt tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag
     3961 cgtcagaccc cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa
     4021 tctgctgctt gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag
     4081 agctaccaac tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg
     4141 tccttctagt gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat
     4201 acctcgctct gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta
     4261 ccgggttgga ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg
     4321 gttcgtgcac acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc
     4381 gtgagctatg agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa
     4441 gcggcagggt cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc
     4501 tttatagtcc tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt
     4561 caggggggcg gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct
     4621 tttgctggcc ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc
     4681 gtattaccgc ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg
     4741 agtcagtgag cgaggaagcg gaagagcgcc tgatgcggta ttttctcctt acgcatctgt
     4801 gcggtatttc acaccgcaat ggtgcactct cagtacaatc tgctctgatg ccgcatagtt
     4861 aagccagtat acactccgct atcgctacgt gactgggtca tggctgcgcc ccgacacccg
     4921 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     4981 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     5041 gcgaggcagc tgcggtaaag ctcatcagcg tggtcgtgaa gcgattcaca gatgtctgcc
     5101 tgttcatccg cgtccagctc gttgagtttc tccagaagcg ttaatgtctg gcttctgata
     5161 aagcgggcca tgttaagggc ggttttttcc tgtttggtca ctgatgcctc cgtgtaaggg
     5221 ggatttctgt tcatgggggt aatgataccg atgaaacgag agaggatgct cacgatacgg
     5281 gttactgatg atgaacatgc ccggttactg gaacgttgtg agggtaaaca actggcggta
     5341 tggatgcggc gggaccagag aaaaatcact cagggtcaat gccagcgctt cgttaataca
     5401 gatgtaggtg ttccacaggg tagccagcag catcctgcga tgcagatccg gaacataatg
     5461 gtgcagggcg ctgacttccg cgtttccaga ctttacgaaa cacggaaacc gaagaccatt
     5521 catgttgttg ctcaggtcgc agacgttttg cagcagcagt cgcttcacgt tcgctcgcgt
     5581 atcggtgatt cattctgcta accagtaagg caaccccgcc agcctagccg ggtcctcaac
     5641 gacaggagca cgatcatgcg cacccgtggc caggacccaa cgctgcccga gatgcgccgc
     5701 gtgcggctgc tggagatggc ggacgcgatg gatatgttct gccaagggtt ggtttgcgca
     5761 ttcacagttc tccgcaagaa ttgattggct ccaattcttg gagtggtgaa tccgttagcg
     5821 aggtgccgcc ggcttccatt caggtcgagg tggcccggct ccatgcaccg cgacgcaacg
     5881 cggggaggca gacaaggtat agggcggcgc ctacaatcca tgccaacccg ttccatgtgc
     5941 tcgccgaggc ggcataaatc gccgtgacga tcagcggtcc aatgatcgaa gttaggctgg
     6001 taagagccgc gagcgatcct tgaagctgtc cctgatggtc gtcatctacc tgcctggaca
     6061 gcatggcctg caacgcgggc atcccgatgc cgccggaagc gagaagaatc ataatgggga
     6121 aggccatcca gcctcgcgtc gcgaacgcca gcaagacgta gcccagcgcg tcggccgcca
     6181 tgccggcgat aatggcctgc ttctcgccga aacgtttggt ggcgggacca gtgacgaagg
     6241 cttgagcgag ggcgtgcaag attccgaata ccgcaagcga caggccgatc atcgtcgcgc
     6301 tccagcgaaa gcggtcctcg ccgaaaatga cccagagcgc tgccggcacc tgtcctacga
     6361 gttgcatgat aaagaagaca gtcataagtg cggcgacgat agtcatgccc cgcgcccacc
     6421 ggaaggagct gactgggttg aaggctctca agggcatcgg tcgagatccc ggtgcctaat
     6481 gagtgagcta acttacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     6541 tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg
     6601 ggcgccaggg tggtttttct tttcaccagt gagacgggca acagctgatt gcccttcacc
     6661 gcctggccct gagagagttg cagcaagcgg tccacgctgg tttgccccag caggcgaaaa
     6721 tcctgtttga tggtggttaa cggcgggata taacatgagc tgtcttcggt atcgtcgtat
     6781 cccactaccg agatatccgc accaacgcgc agcccggact cggtaatggc gcgcattgcg
     6841 cccagcgcca tctgatcgtt ggcaaccagc atcgcagtgg gaacgatgcc ctcattcagc
     6901 atttgcatgg tttgttgaaa accggacatg gcactccagt cgccttcccg ttccgctatc
     6961 ggctgaattt gattgcgagt gagatattta tgccagccag ccagacgcag acgcgccgag
     7021 acagaactta atgggcccgc taacagcgcg atttgctggt gacccaatgc gaccagatgc
     7081 tccacgccca gtcgcgtacc gtcttcatgg gagaaaataa tactgttgat gggtgtctgg
     7141 tcagagacat caagaaataa cgccggaaca ttagtgcagg cagcttccac agcaatggca
     7201 tcctggtcat ccagcggata gttaatgatc agcccactga cgcgttgcgc gagaagattg
     7261 tgcaccgccg ctttacaggc ttcgacgccg cttcgttcta ccatcgacac caccacgctg
     7321 gcacccagtt gatcggcgcg agatttaatc gccgcgacaa tttgcgacgg cgcgtgcagg
     7381 gccagactgg aggtggcaac gccaatcagc aacgactgtt tgcccgccag ttgttgtgcc
     7441 acgcggttgg gaatgtaatt cagctccgcc atcgccgctt ccactttttc ccgcgttttc
     7501 gcagaaacgt ggctggcctg gttcaccacg cgggaaacgg tctgataaga gacaccggca
     7561 tactctgcga catcgtataa cgttactggt ttcacattca ccaccctgaa ttgactctct
     7621 tccgggcgct atcatgccat accgcgaaag gttttgcgcc attcgatggt gtccgggatc
     7681 tcgacgctct cccttatgcg actcctgcat taggaagcag cccagtagta ggttgaggcc
     7741 gttgagcacc gccgccgcaa ggaatggtgc atgcaaggag atggcgccca acagtccccc
     7801 ggccacgggg cctgccacca tacccacgcc gaaacaagcg ctcatgagcc cgaagtggcg
     7861 agcccgatct tccccatcgg tgatgtcggc gatataggcg ccagcaaccg cacctgtggc
     7921 gccggtgatg ccggccacga tgcgtccggc gtagaggatc gagatctcga tcccgcgaaa
     7981 ttaatacgac tcactatagg
//
