LOCUS       sequence-261596-        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"
     primer_bind     53..72
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(92..169)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     375..394
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     478..497
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        478..496
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    497..521
                     /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             536..558
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             577..594
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             604..621
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(634..657,658..693,694..717)
                     /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             718..1011
                     /codon_start=1
                     /gene="S. cerevisiae SMT3 (truncated)"
                     /product="cleavable ubiquitin-like protein tag"
                     /label=SUMO
                     /translation="MSDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPL
                     RRLMEAFAKRQGKEMDSLRFLYDGIRIQADQTPEDLDMEDNDIIEAHREQIGG"
     primer_bind     complement(2978..2996)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2992..3039
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(3214..3219,3220..3236,3237..3242))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(3269..3287)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3355..3459
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3460..3528,3529..4320)
                     /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(3678..3697)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4491..5079
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4980..4999
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5233..5250
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5265..5404
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5330..5349)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5490..5512
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5506..5697)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(7009..8000,1..91))
                     /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 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
       61 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
      121 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
      181 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
      241 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
      301 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
      361 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
      421 gtgatgccgg ccacgatgcg tccggcgtag aggatcgaga tctcgatccc gcgaaattaa
      481 tacgactcac tataggggaa ttgtgagcgg ataacaattc ccctctagaa ataattttgt
      541 ttaactttaa gaaggagata taccatgggc agcagccatc atcatcatca tcacagcagc
      601 ggcctggtgc cgcgcggcag ccatatggct agctggagcc atccgcagtt tgaaaaaggt
      661 ggtggtagcg gtggtggttc aggtggtagt gcatggtcac accctcagtt tgagaaaatg
      721 tcggactcag aagtcaatca agaagctaag ccagaggtca agccagaagt caagcctgag
      781 actcacatca atttaaaggt gtccgatgga tcttcagaga tcttcttcaa gatcaaaaag
      841 accactcctt taagaaggct gatggaagcg ttcgctaaaa gacagggtaa ggaaatggac
      901 tccttaagat tcttgtacga cggtattaga atccaagctg atcagacccc tgaagatttg
      961 gacatggagg ataacgatat tattgaggct cacagagaac agattggtgg atcaagccag
     1021 atcaccatcc aggccagact gatcagcttc gagagcaaca gacagcagct gtggaagctg
     1081 atggccgacc tgaacacccc tctgatcaac gagctgctgt gtcagctggg acagcacccc
     1141 gatttcgaga agtggcagca gaagggcaag ctgcctagca ccgtggtgtc tcagctgtgc
     1201 cagcctctga aaacagaccc cagatttgcc ggccagccta gcagactgta catgagcgcc
     1261 atccacatcg tggactacat ctacaagagc tggctggcca tccagaagcg gctgcagcaa
     1321 cagctggatg gcaagaccag atggctggaa atgctgaaca gcgacgccga gctggtggaa
     1381 ctgtctggcg atacactgga agccatcaga gtgaaggccg ccgagatcct ggccattgct
     1441 atgcctgcca gcgagagcga tagcgctagc cccaaaggca aaaagggcaa gaaagagaag
     1501 aagccctcca gcagcagccc caagagaagc ctgagcaaga ccctgttcga cgcctaccaa
     1561 gagacagagg acatcaagag cagaagcgcc atctcctacc tgctgaagaa cggctgcaag
     1621 ctgaccgaca aagaagagga cagcgagaag ttcgccaagc ggcggagaca ggtggaaatc
     1681 cagatccaga gactgaccga gaagctgatt agcagaatgc ccaagggccg cgacctgacc
     1741 aatgccaagt ggctcgaaac actgctgacc gccacaacca cagtggccga ggataatgcc
     1801 caggccaaga gatggcagga catcctgctg accagaagca gctccctgcc ttttccactg
     1861 gtgttcgaga caaacgagga catggtctgg tccaagaacc agaaaggccg gctgtgcgtg
     1921 cacttcaacg gcctgagcga tctgatcttc gaggtgtact gcggcaaccg gcagctgcac
     1981 tggttccagc ggtttctgga agatcagcag accaagcgca agagcaagaa tcagcacagc
     2041 agcggcctgt tcaccctgag aaatggacac ctcgtgtggc tggaaggcga aggcaaaggc
     2101 gagccttgga acctgcacca cctgacactg tactgctgcg tggacaaccg gctgtggaca
     2161 gaagagggca cagagatcgt gcggcaagag aaggccgatg agatcaccaa gttcatcacc
     2221 aacatgaaga agaagtccga cctgagcgac acccagcagg ccctgattca gagaaagcag
     2281 agcaccctga ccaggatcaa caactccttc gagaggccca gccagccact gtatcagggc
     2341 cagtctcata tcctcgtggg cgtgtcactg ggcctcgaga aacctgctac agtcgccgtg
     2401 gtggacgcca ttgccaacaa ggttctggcc taccggtcca tcaaacagct gctcggcgac
     2461 aactatgagc tgctgaaccg tcagcgacgg cagcagcagt atctgagcca cgagagacac
     2521 aaggcccaga agaacttcag ccccaaccag tttggcgcct ctgaactggg ccagcacatc
     2581 gatagactgc tggccaaggc cattgtggcc ctggccagaa catacaaggc cggcagtatc
     2641 gtgctgccca agctgggcga tatgagagaa gtggtgcaga gcgagatcca ggctatcgcc
     2701 gaacagaagt tccccggcta tatcgagggc cagcagaagt atgccaagca gtatagagtg
     2761 aacgtgcacc ggtggtccta cggcaggctg atccagagca tccagtctaa ggctgcccag
     2821 accggcatcg tgatcgagga aggcaagcag cccattagag gcagccctca cgacaaggct
     2881 aaagaactgg ctctgagcgc ctacaacctg aggctgacaa gaagaagctg agcggccgca
     2941 ctcgaggccc gaaaggaagc tgagttggct gctgccaccg ctgagcaata actagcataa
     3001 ccccttgggg cctctaaacg ggtcttgagg ggttttttgc tgaaaggagg aactatatcc
     3061 ggatatcccg caagaggccc ggcagtaccg gcataaccaa gcctatgcct acagcatcca
     3121 gggtgacggt gccgaggatg acgatgagcg cattgttaga tttcatacac ggtgcctgac
     3181 tgcgttagca atttaactgt gataaactac cgcattaaag cttatcgatg ataagctgtc
     3241 aaacatgaga attcttgaag acgaaagggc ctcgtgatac gcctattttt ataggttaat
     3301 gtcatgataa taatggtttc ttagacgtca ggtggcactt ttcggggaaa tgtgcgcgga
     3361 acccctattt gtttattttt ctaaatacat tcaaatatgt atccgctcat gagacaataa
     3421 ccctgataaa tgcttcaata atattgaaaa aggaagagta tgagtattca acatttccgt
     3481 gtcgccctta ttcccttttt tgcggcattt tgccttcctg tttttgctca cccagaaacg
     3541 ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac gagtgggtta catcgaactg
     3601 gatctcaaca gcggtaagat ccttgagagt tttcgccccg aagaacgttt tccaatgatg
     3661 agcactttta aagttctgct atgtggcgcg gtattatccc gtgttgacgc cgggcaagag
     3721 caactcggtc gccgcataca ctattctcag aatgacttgg ttgagtactc accagtcaca
     3781 gaaaagcatc ttacggatgg catgacagta agagaattat gcagtgctgc cataaccatg
     3841 agtgataaca ctgcggccaa cttacttctg acaacgatcg gaggaccgaa ggagctaacc
     3901 gcttttttgc acaacatggg ggatcatgta actcgccttg atcgttggga accggagctg
     3961 aatgaagcca taccaaacga cgagcgtgac accacgatgc ctgcagcaat ggcaacaacg
     4021 ttgcgcaaac tattaactgg cgaactactt actctagctt cccggcaaca attaatagac
     4081 tggatggagg cggataaagt tgcaggacca cttctgcgct cggcccttcc ggctggctgg
     4141 tttattgctg ataaatctgg agccggtgag cgtgggtctc gcggtatcat tgcagcactg
     4201 gggccagatg gtaagccctc ccgtatcgta gttatctaca cgacggggag tcaggcaact
     4261 atggatgaac gaaatagaca gatcgctgag ataggtgcct cactgattaa gcattggtaa
     4321 ctgtcagacc aagtttactc atatatactt tagattgatt taaaacttca tttttaattt
     4381 aaaaggatct aggtgaagat cctttttgat aatctcatga ccaaaatccc ttaacgtgag
     4441 ttttcgttcc actgagcgtc agaccccgta gaaaagatca aaggatcttc ttgagatcct
     4501 ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac caccgctacc agcggtggtt
     4561 tgtttgccgg atcaagagct accaactctt tttccgaagg taactggctt cagcagagcg
     4621 cagataccaa atactgtcct tctagtgtag ccgtagttag gccaccactt caagaactct
     4681 gtagcaccgc ctacatacct cgctctgcta atcctgttac cagtggctgc tgccagtggc
     4741 gataagtcgt gtcttaccgg gttggactca agacgatagt taccggataa ggcgcagcgg
     4801 tcgggctgaa cggggggttc gtgcacacag cccagcttgg agcgaacgac ctacaccgaa
     4861 ctgagatacc tacagcgtga gctatgagaa agcgccacgc ttcccgaagg gagaaaggcg
     4921 gacaggtatc cggtaagcgg cagggtcgga acaggagagc gcacgaggga gcttccaggg
     4981 ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc acctctgact tgagcgtcga
     5041 tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa acgccagcaa cgcggccttt
     5101 ttacggttcc tggccttttg ctggcctttt gctcacatgt tctttcctgc gttatcccct
     5161 gattctgtgg ataaccgtat taccgccttt gagtgagctg ataccgctcg ccgcagccga
     5221 acgaccgagc gcagcgagtc agtgagcgag gaagcggaag agcgcctgat gcggtatttt
     5281 ctccttacgc atctgtgcgg tatttcacac cgcaatggtg cactctcagt acaatctgct
     5341 ctgatgccgc atagttaagc cagtatacac tccgctatcg ctacgtgact gggtcatggc
     5401 tgcgccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc
     5461 atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc
     5521 gtcatcaccg aaacgcgcga ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga
     5581 ttcacagatg tctgcctgtt catccgcgtc cagctcgttg agtttctcca gaagcgttaa
     5641 tgtctggctt ctgataaagc gggccatgtt aagggcggtt ttttcctgtt tggtcactga
     5701 tgcctccgtg taagggggat ttctgttcat gggggtaatg ataccgatga aacgagagag
     5761 gatgctcacg atacgggtta ctgatgatga acatgcccgg ttactggaac gttgtgaggg
     5821 taaacaactg gcggtatgga tgcggcggga ccagagaaaa atcactcagg gtcaatgcca
     5881 gcgcttcgtt aatacagatg taggtgttcc acagggtagc cagcagcatc ctgcgatgca
     5941 gatccggaac ataatggtgc agggcgctga cttccgcgtt tccagacttt acgaaacacg
     6001 gaaaccgaag accattcatg ttgttgctca ggtcgcagac gttttgcagc agcagtcgct
     6061 tcacgttcgc tcgcgtatcg gtgattcatt ctgctaacca gtaaggcaac cccgccagcc
     6121 tagccgggtc ctcaacgaca ggagcacgat catgcgcacc cgtggccagg acccaacgct
     6181 gcccgagatg cgccgcgtgc ggctgctgga gatggcggac gcgatggata tgttctgcca
     6241 agggttggtt tgcgcattca cagttctccg caagaattga ttggctccaa ttcttggagt
     6301 ggtgaatccg ttagcgaggt gccgccggct tccattcagg tcgaggtggc ccggctccat
     6361 gcaccgcgac gcaacgcggg gaggcagaca aggtataggg cggcgcctac aatccatgcc
     6421 aacccgttcc atgtgctcgc cgaggcggca taaatcgccg tgacgatcag cggtccaatg
     6481 atcgaagtta ggctggtaag agccgcgagc gatccttgaa gctgtccctg atggtcgtca
     6541 tctacctgcc tggacagcat ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga
     6601 agaatcataa tggggaaggc catccagcct cgcgtcgcga acgccagcaa gacgtagccc
     6661 agcgcgtcgg ccgccatgcc ggcgataatg gcctgcttct cgccgaaacg tttggtggcg
     6721 ggaccagtga cgaaggcttg agcgagggcg tgcaagattc cgaataccgc aagcgacagg
     6781 ccgatcatcg tcgcgctcca gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc
     6841 ggcacctgtc ctacgagttg catgataaag aagacagtca taagtgcggc gacgatagtc
     6901 atgccccgcg cccaccggaa ggagctgact gggttgaagg ctctcaaggg catcggtcga
     6961 gatcccggtg cctaatgagt gagctaactt acattaattg cgttgcgctc actgcccgct
     7021 ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg cgcggggaga
     7081 ggcggtttgc gtattgggcg ccagggtggt ttttcttttc accagtgaga cgggcaacag
     7141 ctgattgccc ttcaccgcct ggccctgaga gagttgcagc aagcggtcca cgctggtttg
     7201 ccccagcagg cgaaaatcct gtttgatggt ggttaacggc gggatataac atgagctgtc
     7261 ttcggtatcg tcgtatccca ctaccgagat atccgcacca acgcgcagcc cggactcggt
     7321 aatggcgcgc attgcgccca gcgccatctg atcgttggca accagcatcg cagtgggaac
     7381 gatgccctca ttcagcattt gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc
     7441 ttcccgttcc gctatcggct gaatttgatt gcgagtgaga tatttatgcc agccagccag
     7501 acgcagacgc gccgagacag aacttaatgg gcccgctaac agcgcgattt gctggtgacc
     7561 caatgcgacc agatgctcca cgcccagtcg cgtaccgtct tcatgggaga aaataatact
     7621 gttgatgggt gtctggtcag agacatcaag aaataacgcc ggaacattag tgcaggcagc
     7681 ttccacagca atggcatcct ggtcatccag cggatagtta atgatcagcc cactgacgcg
     7741 ttgcgcgaga agattgtgca ccgccgcttt acaggcttcg acgccgcttc gttctaccat
     7801 cgacaccacc acgctggcac ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg
     7861 cgacggcgcg tgcagggcca gactggaggt ggcaacgcca atcagcaacg actgtttgcc
     7921 cgccagttgt tgtgccacgc ggttgggaat gtaattcagc tccgccatcg ccgcttccac
     7981 tttttcccgc gttttcgcag
//
