LOCUS       sequence-345509-        6490 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Bacterial expression of KraIscB-1-TwinStrep.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6490)
  AUTHORS   Altae-Tran H, Kannan S, Demircioglu FE, Oshiro R, Nety SP, McKay LJ,
            Dlakic M, Inskeep WP, Makarova KS, Macrae RK, Koonin EV, Zhang F
  TITLE     The widespread IS200/IS605 transposon family encodes diverse 
            programmable RNA-guided endonucleases.
  JOURNAL   Science. 2021 Oct;374(6563):57-65. doi: 10.1126/science.abj6856. 
            Epub 2021 Sep 9.
  PUBMED    34591643
REFERENCE   2  (bases 1 to 6490)
  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.abj6856"; journalName: "Science"; date: "2021-10"; 
            volume: "374"; issue: "6563"; pages: "57-65"
COMMENT     Sequence Label: pHS0663 pET45b(+) KraIscB-1 TwinStrep
FEATURES             Location/Qualifiers
     source          1..6490
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     951..970
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(990..1067)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     1273..1292
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     primer_bind     1380..1399
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1380..1398
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1399..1423
                     /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             1438..1460
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             2778..2795
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             join(2808..2831,2832..2867,2868..2891)
                     /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"
     primer_bind     complement(2917..2935)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      2931..2978
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     rep_origin      3015..3470
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3102..3121)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3312..3333
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             complement(join(3585..4376,4377..4445))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     4208..4227
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4446..4538)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4619..5207
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5108..5127
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5361..5378
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    5393..5532
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(5458..5477)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     5618..5640
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(5634..5825)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(join(6397..6490,1..989))
                     /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 agggtggttt ttcttttcac cagtgagacg ggcaacagct gattgccctt caccgcctgg
       61 ccctgagaga gttgcagcaa gcggtccacg ctggtttgcc ccagcaggcg aaaatcctgt
      121 ttgatggtgg ttaacggcgg gatataacat gagctgtctt cggtatcgtc gtatcccact
      181 accgagatgt ccgcaccaac gcgcagcccg gactcggtaa tggcgcgcat tgcgcccagc
      241 gccatctgat cgttggcaac cagcatcgca gtgggaacga tgccctcatt cagcatttgc
      301 atggtttgtt gaaaaccgga catggcactc cagtcgcctt cccgttccgc tatcggctga
      361 atttgattgc gagtgagata tttatgccag ccagccagac gcagacgcgc cgagacagaa
      421 cttaatgggc ccgctaacag cgcgatttgc tggtgaccca atgcgaccag atgctccacg
      481 cccagtcgcg taccgtcttc atgggagaaa ataatactgt tgatgggtgt ctggtcagag
      541 acatcaagaa ataacgccgg aacattagtg caggcagctt ccacagcaat ggcatcctgg
      601 tcatccagcg gatagttaat gatcagccca ctgacgcgtt gcgcgagaag attgtgcacc
      661 gccgctttac aggcttcgac gccgcttcgt tctaccatcg acaccaccac gctggcaccc
      721 agttgatcgg cgcgagattt aatcgccgcg acaatttgcg acggcgcgtg cagggccaga
      781 ctggaggtgg caacgccaat cagcaacgac tgtttgcccg ccagttgttg tgccacgcgg
      841 ttgggaatgt aattcagctc cgccatcgcc gcttccactt tttcccgcgt tttcgcagaa
      901 acgtggctgg cctggttcac cacgcgggaa acggtctgat aagagacacc ggcatactct
      961 gcgacatcgt ataacgttac tggtttcaca ttcaccaccc tgaattgact ctcttccggg
     1021 cgctatcatg ccataccgcg aaaggttttg cgccattcga tggtgtccgg gatctcgacg
     1081 ctctccctta tgcgactcct gcattaggaa gcagcccagt agtaggttga ggccgttgag
     1141 caccgccgcc gcaaggaatg gtgcatgcaa ggagatggcg cccaacagtc ccccggccac
     1201 ggggcctgcc accataccca cgccgaaaca agcgctcatg agcccgaagt ggcgagcccg
     1261 atcttcccca tcggtgatgt cggcgatata ggcgccagca accgcacctg tggcgccggt
     1321 gatgccggcc acgatgcgtc cggcgtagag gatcgagatc gatctcgatc ccgcgaaatt
     1381 aatacgactc actatagggg aattgtgagc ggataacaat tcccctctag aaataatttt
     1441 gtttaacttt aagaaggaga tataccatga gcaagaacgt cgtctacgtc ctctcgccgg
     1501 agagaacacc attaatgccc tgtcaacctg ccattgcgag gttgttgctg aaacaaggaa
     1561 aagcaaaggt gaggcatcga acgcccttta cgattcagct tctcgcacag ccagagcacg
     1621 tgtacacaca accgctgacc catggcgttg atacgggaag ttccataatc ggatcagccg
     1681 tggctaatga gcatggacac gtcgtgtatc tttcggaagt cgagatacgc aatgatattg
     1741 caaacactat gaaggaacga gcgagagcac gccgcaatcg tcgtcaacgc aagacacgct
     1801 atcgccctgc tcgctggctc aatcgcaaga aatcgatcaa aactggacgc ttctcgccca
     1861 ccatgagaag caagattgat actcatctgc gagaaattcg ctttatacgg tccttgctgc
     1921 ccatcacgtc tacgatacta gaaacaggct catttgatcc ttatgcactc agaaatcctg
     1981 aagtcctgca aaagaagtgg ctctaccaga ggggcatcaa ctacggtttt gccaatacca
     2041 aagcctatgt gctcacacga gacggctacc tctgtcagca gtgcaaaggg aagtcaaagg
     2101 accgacggct tgaagttcac cacatcatct tcagaagtcg aaatggaagc gatgaggaag
     2161 cgaatttact cactctctgc aaaacttgtc atgatggact ccatgcaggc accatcaccc
     2221 tgaaactcac aggcaagaaa aagggaacct tgcaacatgc gacccagatg aatagcatcc
     2281 gcattcagtt actcaagcgt gttgaggcag aggaaacctg gggctttgtc accaaagagc
     2341 atcgtcttct ggtaggactc cccaaagagc atatctttga tgcagccgtg attgcaacac
     2401 gaggagtgaa gccaaccttc tataccacga gcgtgctctc aaaacactgt gtgtcagatg
     2461 gagattacaa acaaacgaaa ggaaaacacg gtcaacaacg agtgaacaca ggtaagatca
     2521 tgggatttcg caagtttgat aaggtgtact atttggggaa ggagtacttt atcaagggga
     2581 gaatgtctac cggctacgcg atcctcatgg acattgacgg caacaaaatt gagttcaaac
     2641 cacttcccaa gtttgacaaa atgaagagag taagtgcacg ttcatcatgg atgatgaaac
     2701 aaagaaccac gccaaatccc tcattctcta tcacctcatc tttgtctgca agtgcgggaa
     2761 aaaacgttag cagcggcctg gtgccgcgcg gcagccatat ggctagctgg agccatccgc
     2821 agtttgaaaa aggtggtggt agcggtggtg gttcaggtgg tagtgcatgg tcacaccctc
     2881 agtttgagaa aaattaatta acctaggctg ctgccaccgc tgagcaataa ctagcataac
     2941 cccttggggc ctctaaacgg gtcttgaggg gttttttgct gaaaggagga actatatccg
     3001 gattggcgaa tgggacgcgc cctgtagcgg cgcattaagc gcggcgggtg tggtggttac
     3061 gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg ctttcttccc
     3121 ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg ggctcccttt
     3181 agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt agggtgatgg
     3241 ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt tggagtccac
     3301 gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta tctcggtcta
     3361 ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa atgagctgat
     3421 ttaacaaaaa tttaacgcga attttaacaa aatattaacg tttacaattt ctggcggcac
     3481 gatggcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     3541 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     3601 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     3661 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     3721 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     3781 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     3841 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     3901 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     3961 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     4021 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     4081 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     4141 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     4201 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     4261 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     4321 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     4381 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     4441 ctcatactct tcctttttca atcatgattg aagcatttat cagggttatt gtctcatgag
     4501 cggatacata tttgaatgta tttagaaaaa taaacaaata ggtcatgacc aaaatccctt
     4561 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt
     4621 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag
     4681 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca
     4741 gcagagcgca gataccaaat actgtccttc tagtgtagcc gtagttaggc caccacttca
     4801 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg
     4861 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg
     4921 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct
     4981 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga
     5041 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc
     5101 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg
     5161 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg
     5221 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt
     5281 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc
     5341 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcctgatgc
     5401 ggtattttct ccttacgcat ctgtgcggta tttcacaccg caatggtgca ctctcagtac
     5461 aatctgctct gatgccgcat agttaagcca gtatacactc cgctatcgct acgtgactgg
     5521 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     5581 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     5641 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     5701 tgaagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     5761 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     5821 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     5881 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     5941 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     6001 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     6061 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     6121 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     6181 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     6241 cgccagccta gccgggtcct caacgacagg agcacgatca tgctagtcat gccccgcgcc
     6301 caccggaagg agctgactgg gttgaaggct ctcaagggca tcggtcgaga tcccggtgcc
     6361 taatgagtga gctaacttac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga
     6421 aacctgtcgt gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt
     6481 attgggcgcc
//
