LOCUS       sequence-355130-        7521 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of X. albilineans type I-C cas3.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7521)
  AUTHORS   Wimmer F, Mougiakos I, Englert F, Beisel CL
  TITLE     Rapid cell-free characterization of multi-subunit CRISPR effectors 
            and transposons.
  JOURNAL   Mol Cell. 2022 Feb 16. pii: S1097-2765(22)00102-2. doi: 
            10.1016/j.molcel.2022.01.026.
  PUBMED    35216669
REFERENCE   2  (bases 1 to 7521)
  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"; journalName: "Mol Cell. 
            2022 Feb 16. pii: S1097-2765(22)00102-2. doi: 
            10.1016/j.molcel.2022.01.026."
COMMENT     Sequence Label: pXalb_IC_Cas3
FEATURES             Location/Qualifiers
     source          1..7521
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     terminator      complement(26..73)
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     primer_bind     69..87
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     CDS             complement(140..157)
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     CDS             complement(1865..1876)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     RBS             2462..2484
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     protein_bind    2499..2523
                     /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)."
     primer_bind     complement(2523..2542)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2524..2542)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2626..2645)
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2851..2928
                     /gene="lacI"
                     /label=lacI promoter
     CDS             2929..4011
                     /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     complement(2948..2967)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     CDS             4820..5011
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     primer_bind     complement(5005..5027)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     misc_feature    5113..5252
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     5168..5187
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     complement(5267..5284)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5438..6026)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5518..5537)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     complement(6137..6156)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             6148..6963
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             6148..6963
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(6221..6240)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      complement(7055..7510)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(7192..7213)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     7404..7423
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 atccggatat agttcctcct ttcagcaaaa aacccctcaa gacccgttta gaggccccaa
       61 ggggttatgc tagttattgc tcagcggtgg cagcagccaa ctcagcttcc tttcgggctt
      121 tgttagcagc cggatctcag tggtggtggt ggtggtgcca aaaaagctgc tctccctcca
      181 aaaactgcgg gttgtcccaa tgcaacccga accgggcgtc gtacaggcgt ggattacaca
      241 ggcgcacgaa ttgctcctcg ccgcagcgct cgggtgcgac cggggcgata gcgtgcgcct
      301 gccagagcgc cttgtaggcc tgctcgggca cctggaccag ccaggattgc aggcggcggg
      361 cggcaccggc cacgctgaca cgctcggcat gctcaagctc gtgcaggact tcggcgacct
      421 cgggcacttc cttgtccgac tccatcgcgt agggggcgta aggcacgatc aatggacgca
      481 tggtggtctg gatcacgttg aacttgcgcg caagtgtgtt caggggaaga ttgtcgatgg
      541 tggacttgcg cagtaggctc aggattgcct cgttgtcgag tctgtcatcg ccaagacgat
      601 gatagagttt gcgaaagtag ctgtggatgg cgtccattgc cagcaggtcg ccgtgatgct
      661 cgcgctcgac atcgcggaag acttcggcga acagttccag ttccctcggt ggcttccagt
      721 ttgtgttggt tggcgcgaag acgcgtactt cgctgctgtc gatgtcccac tccatgttgc
      781 gattgcagcg accagcggcc tgggcaatgg aatccagtcc tgcttcggcg cgtagcacca
      841 gcggtacgct cagatctatc ccagcttcaa tcaaactggt cgagacaatc cggcatggcc
      901 ggccttcctg caaatctttt cgtatctgtg cgagcaccac acggcgatgc cgcgcatgca
      961 tcagcgtggt cagatggcgt gcgcccggct gatcggcgat tgcgttgaac aaggcatgtg
     1021 cgtggcgacg gttgttgacg atgcatagca cctgctcgcg ttgccggagc tgatcggcca
     1081 gcgcatcgtc gtccagaggg ccgacatgcc gcacgcgtac acggcgcagt tgcgtgtaca
     1141 gcgcctgcgg ttcattcgcc agttcctcca cctgttcgag gccgtcggcg aagccctggt
     1201 cctggcgcag cgcgggttgg gtggcggtac acagcaccgg gctgacccga tagttgagcg
     1261 ccagctcgtc cagcagcgcc acacacgggc gcaataattt gtatggcagg gtctgtgctt
     1321 cgtccaggat caccacgctg ccggggatgt tgtgcagctt gcggcagcgc gaggggcggt
     1381 cggtaaacag gctttcgaaa aactgcacgg cggtggtgac cacgatcggc gcgtcccagt
     1441 tgtccatcgc cagtttgcgc ttttcgatcg cctgcggcgc cttgctcgga tcgtcgaaga
     1501 aggcgctgtg atgttccagc accacagcgc tgttgtcctg cgcgccgagc gcgtgacgga
     1561 agacgctggc ggtctggtcg acgatgctgg tgaacggaat gacgtagatc acccggcgca
     1621 gcccgtgcac gatggcgtga tccaacgcga atgccagcga tgccagggtc ttgccgccgc
     1681 cggtcggcac ggtgagcgag aacaggccag tgcgcagatg ggccttggcg cgcacgtcgt
     1741 gcagaatctg cgcccgtacc ggattgatgc cacctttgct cgggaggcca gccagatgcg
     1801 tgtcgaggcg ttcgcgcagt tctgccagtg tcggctgcgc acccacttct tcttcgcgtg
     1861 tcttgcggcc ttcgatgcgc cggtagaagt cgtcggtatc gacgaaatcg gcatccacta
     1921 gacaggagaa gatcatccgt gccagtagcg tcatctggaa ggcgcggcgt tctggacggg
     1981 gatggcgggc gaagccctgc ggaaattcca gcttggccgg cagtgtcagt tcgcgctccc
     2041 agtcaggtaa taagggaggc aacgcctgtg cgcgatactc ctccgacagc cgttcgtgta
     2101 gcgaggtgcg agaggagtct ttgtcctggc gtccgtccgc cagcccggcg tgatgtccag
     2161 cgatggtgta ggccagaagt ttcccaccat cgccataacg cgcacaagca atccgcgccc
     2221 cccaagtggc gtgatccacc ttggcggggt cgccgtctag gcgcgcctgg aatttctcgg
     2281 tgtatttgcc gagatcgtgc aattgcccag cgacccgggc cagcagttgt ccgccgaagg
     2341 gcccggcgtg gcgcgcggca cgctcgccga cgccgttcag atgatcggcc agaccttgcc
     2401 aattgctacg atcgctgcgt tcggtcgaat gcgcatagaa cgccatcgtg tctcccgtat
     2461 atctccttct taaagttaaa caaaattatt tctagagggg aattgttatc cgctcacaat
     2521 tcccctatag tgagtcgtat taatttcgcg ggatcgagat ctcgatcctc tacgccggac
     2581 gcatcgtggc cggcatcacc ggcgccacag gtgcggttgc tggcgcctat atcgccgaca
     2641 tcaccgatgg ggaagatcgg gctcgccact tcgggctcat gagcgcttgt ttcggcgtgg
     2701 gtatggtggc aggccccgtg gccgggggac tgttgggcgc catctccttg catgcaccat
     2761 tccttgcggc ggcggtgctc aacggcctca acctactact gggctgcttc ctaatgcagg
     2821 agtcgcataa gggagagcgt cgagatcccg gacaccatcg aatggcgcaa aacctttcgc
     2881 ggtatggcat gatagcgccc ggaagagagt caattcaggg tggtgaatgt gaaaccagta
     2941 acgttatacg atgtcgcaga gtatgccggt gtctcttatc agaccgtttc ccgcgtggtg
     3001 aaccaggcca gccacgtttc tgcgaaaacg cgggaaaaag tggaagcggc gatggcggag
     3061 ctgaattaca ttcccaaccg cgtggcacaa caactggcgg gcaaacagtc gttgctgatt
     3121 ggcgttgcca cctccagtct ggccctgcac gcgccgtcgc aaattgtcgc ggcgattaaa
     3181 tctcgcgccg atcaactggg tgccagcgtg gtggtgtcga tggtagaacg aagcggcgtc
     3241 gaagcctgta aagcggcggt gcacaatctt ctcgcgcaac gcgtcagtgg gctgatcatt
     3301 aactatccgc tggatgacca ggatgccatt gctgtggaag ctgcctgcac taatgttccg
     3361 gcgttatttc ttgatgtctc tgaccagaca cccatcaaca gtattatttt ctcccatgaa
     3421 gacggtacgc gactgggcgt ggagcatctg gtcgcattgg gtcaccagca aatcgcgctg
     3481 ttagcgggcc cattaagttc tgtctcggcg cgtctgcgtc tggctggctg gcataaatat
     3541 ctcactcgca atcaaattca gccgatagcg gaacgggaag gcgactggag tgccatgtcc
     3601 ggttttcaac aaaccatgca aatgctgaat gagggcatcg ttcccactgc gatgctggtt
     3661 gccaacgatc agatggcgct gggcgcaatg cgcgccatta ccgagtccgg gctgcgcgtt
     3721 ggtgcggata tctcggtagt gggatacgac gataccgaag acagctcatg ttatatcccg
     3781 ccgttaacca ccatcaaaca ggattttcgc ctgctggggc aaaccagcgt ggaccgcttg
     3841 ctgcaactct ctcagggcca ggcggtgaag ggcaatcagc tgttgcccgt ctcactggtg
     3901 aaaagaaaaa ccaccctggc gcccaatacg caaaccgcct ctccccgcgc gttggccgat
     3961 tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg agcgcaacgc
     4021 aattaatgta agttagctca ctcattaggc accgggatct cgaccgatgc ccttgagagc
     4081 cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat
     4141 gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct gggtcatttt
     4201 cggcgaggac cgctttcgct ggagcgcgac gatgatcggc ctgtcgcttg cggtattcgg
     4261 aatcttgcac gccctcgctc aagccttcgt cactggtccc gccaccaaac gtttcggcga
     4321 gaagcaggcc attatcgccg gcatggcggc cccacgggtg cgcatgatcg tgctcctgtc
     4381 gttgaggacc cggctaggct ggcggggttg ccttactggt tagcagaatg aatcaccgat
     4441 acgcgagcga acgtgaagcg actgctgctg caaaacgtct gcgacctgag caacaacatg
     4501 aatggtcttc ggtttccgtg tttcgtaaag tctggaaacg cggaagtcag cgccctgcac
     4561 cattatgttc cggatctgca tcgcaggatg ctgctggcta ccctgtggaa cacctacatc
     4621 tgtattaacg aagcgctggc attgaccctg agtgattttt ctctggtccc gccgcatcca
     4681 taccgccagt tgtttaccct cacaacgttc cagtaaccgg gcatgttcat catcagtaac
     4741 ccgtatcgtg agcatcctct ctcgtttcat cggtatcatt acccccatga acagaaatcc
     4801 cccttacacg gaggcatcag tgaccaaaca ggaaaaaacc gcccttaaca tggcccgctt
     4861 tatcagaagc cagacattaa cgcttctgga gaaactcaac gagctggacg cggatgaaca
     4921 ggcagacatc tgtgaatcgc ttcacgacca cgctgatgag ctttaccgca gctgcctcgc
     4981 gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc
     5041 ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg
     5101 cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt
     5161 aactatgcgg catcagagca gattgtactg agagtgcacc attgcggtgt gaaataccgc
     5221 acagatgcgt aaggagaaaa taccgcatca ggcgctcttc cgcttcctcg ctcactgact
     5281 cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac
     5341 ggttatccac agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa
     5401 aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg
     5461 acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa
     5521 gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc
     5581 ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac
     5641 gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac
     5701 cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg
     5761 taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt
     5821 atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagga
     5881 cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct
     5941 cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga
     6001 ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg
     6061 ctcagtggaa cgaaaactca cgttaaggga ttttggtcat gaacaataaa actgtctgct
     6121 tacataaaca gtaatacaag gggtgttatg agccatattc aacgggaaac gtcttgctct
     6181 aggccgcgat taaattccaa catggatgct gatttatatg ggtataaatg ggctcgcgat
     6241 aatgtcgggc aatcaggtgc gacaatctat cgattgtatg ggaagcccga tgcgccagag
     6301 ttgtttctga aacatggcaa aggtagcgtt gccaatgatg ttacagatga gatggtcaga
     6361 ctaaactggc tgacggaatt tatgcctctt ccgaccatca agcattttat ccgtactcct
     6421 gatgatgcat ggttactcac cactgcgatc cccgggaaaa cagcattcca ggtattagaa
     6481 gaatatcctg attcaggtga aaatattgtt gatgcgctgg cagtgttcct gcgccggttg
     6541 cattcgattc ctgtttgtaa ttgtcctttt aacagcgatc gcgtatttcg tctcgctcag
     6601 gcgcaatcac gaatgaataa cggtttggtt gatgcgagtg attttgatga cgagcgtaat
     6661 ggctggcctg ttgaacaagt ctggaaagaa atgcataaac ttttgccatt ctcaccggat
     6721 tcagtcgtca ctcatggtga tttctcactt gataacctta tttttgacga ggggaaatta
     6781 ataggttgta ttgatgttgg acgagtcgga atcgcagacc gataccagga tcttgccatc
     6841 ctatggaact gcctcggtga gttttctcct tcattacaga aacggctttt tcaaaaatat
     6901 ggtattgata atcctgatat gaataaattg cagtttcatt tgatgctcga tgagtttttc
     6961 taagaattaa ttcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     7021 ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta
     7081 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     7141 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     7201 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     7261 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     7321 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     7381 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     7441 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     7501 cagggcgcgt cccattcgcc a
//
