LOCUS       sequence-490430-        6198 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  for E.coli plasmid interference and PAM depletion assays.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6198)
  AUTHORS   Jin S, Zhu Z, Li Y, Zhang S, Liu Y, Li D, Li Y, Luo Y, Cheng Z, Zhao
            KT, Gao Q, Yang G, Li H, Liang R, Zhang R, Qiu JL, Zhang YE, Liu JG,
            Gao C
  TITLE     Functional RNA splitting drove the evolutionary emergence of type V 
            CRISPR-Cas systems from transposons.
  JOURNAL   Cell. 2025 Sep 29:S0092-8674(25)01035-9. doi: 
            10.1016/j.cell.2025.09.004.
  PUBMED    41027434
REFERENCE   2  (bases 1 to 6198)
  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: "Cell. 2025 
            Sep 29:S0092-8674(25)01035-9. doi: 10.1016/j.cell.2025.09.004."
COMMENT     Sequence Label: pEffector-ISDra2 TnpB-artificial CRISPR RNA
FEATURES             Location/Qualifiers
     source          1..6198
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             complement(52..867)
                     /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"
     primer_bind     775..794
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     859..878
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     primer_bind     complement(1258..1275)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(1392..1937)
                     /direction=LEFT
                     /label=p15A ori
                     /note="Plasmids containing the medium-copy-number p15A 
                     origin of replication can be propagated in E. coli cells 
                     that contain a second plasmid with the ColE1 origin.
                     "
     primer_bind     complement(1472..1491)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2013..2032
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
     promoter        2186..2220
                     /label=J23119(SpeI) promoter
                     /note="bacterial promoter (Registry of Standard Biological 
                     Parts BBa_J23119) modified to end with an SpeI site"
     CDS             3087..3116
                     /codon_start=1
                     /product="Myc (human c-Myc proto-oncogene) epitope tag"
                     /label=Myc
                     /translation="EQKLISEEDL"
     CDS             3132..3149
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(3205..3222)
                     /label=pBAD Reverse
                     /note="For vectors with E. coli araBAD promoter, reverse 
                     primer"
     terminator      3375..3421
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     CDS             complement(3448..4530)
                     /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     4492..4511
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4665..4670,4671..4686,4687..4693)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    4699..4718
                     /label=lac operator (symmetric)
                     /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). The 
                     symmetric lac operator was optimized for tight binding of 
                     lac repressor."
     misc_RNA        4721..4770
                     /label=sTRSV HHRz
                     /note="hammerhead ribozyme from the tobacco ringspot virus 
                     satellite RNA (Khvorova et al., 2003)"
ORIGIN
        1 cagataaaaa aaatccttag ctttcgctaa ggatgatttc tgctagtatt attagaaaaa
       61 ctcatcgagc atcaaatgaa actgcaattt attcatatca ggattatcaa taccatattt
      121 ttgaaaaagc cgtttctgta atgaaggaga aaactcaccg aggcagttcc aaagaatggc
      181 aaggtcctgg taacggtctg cgattccgac ccgtccaaca tcaatacaac ctattaattt
      241 cccctcgtca aaaataaggt tatcaagtga gaaatcacca tgagtgacga ctgaatccgg
      301 tgagaatggc aagagcttgt gcatttcttt ccagacttgt tcaacaggcc agccattacg
      361 ctcgtcatca aaatcactcg catcaaccaa accgttattc atgcgtgatt gcgcctgagc
      421 aagacgaaat acacgatcgc tgttaaaagg acaattacaa acaggaatcg aatgtaaccg
      481 gcgcaggaac acggccagcg catcaacaat attttcacct gaatcaggat attcttctaa
      541 tacctggaag gctgttttcc caggaatcgc ggtggtgagt aaccacgcat catcaggagt
      601 acggataaaa tgcttgatgg tcgggagagg cataaactcc gtcagccagt tgagacggac
      661 catctcatct gtaacatcat tggcaacgct acctttgcca tgtttcagaa acaactctgg
      721 cgcatcgggc ttcccataca agcgatagat tgtcgcacct gattgcccga cattatcgcg
      781 agcccattta tacccatata aatcagcgtc catgttggag tttaagcgcg gacgggagca
      841 agacgtttcc cgttgaatat ggctcataac accccttgta ttactgttta tgtaagcaga
      901 cagttttatt gttcatgatg atatattttt atcttgtgca atgtaacatc agagattttg
      961 agacacaacg tggctttgtt gaataaatcg aacttttgct gagttgaagg atcagctcta
     1021 gtagttacat tgtcgatctg ttcatggtga acagctttga atgcaccaaa aactcgtaaa
     1081 agctctgatg tatctatctt ttttacaccg ttttcatctg tgcatagcta gcggagtgta
     1141 tactggctta ctatgttggc actgatgagg gtgtcagtga agtgcttcat gtggcaggag
     1201 aaaaaaggct gcaccggtgc gtcagcagaa tatgtgatac aggatatatt ccgcttcctc
     1261 gctcactgac tcgctacgct cggtcgttcg actgcggcga gcggaaatgg cttacgaacg
     1321 gggcggagat ttcctggaag atgccaggaa gatacttaac agggaagtga gagggccgcg
     1381 gcaaagccgt ttttccatag gctccgcccc cctgacaagc atcacgaaat ctgacgctca
     1441 aatcagtggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggcggc
     1501 tccctcgtgc gctctcctgt tcctgccttt cggtttaccg gtgtcattcc gctgttatgg
     1561 ccgcgtttgt ctcattccac gcctgacact cagttccggg taggcagttc gctccaagct
     1621 ggactgtatg cacgaacccc ccgttcagtc cgaccgctgc gccttatccg gtaactatcg
     1681 tcttgagtcc aacccggaaa gacatgcaaa agcaccactg gcagcagcca ctggtaattg
     1741 atttagagga gttagtcttg aagtcatgcg ccggttaagg ctaaactgaa aggacaagtt
     1801 ttggtgactg cgctcctcca agccagttac ctcggttcaa agagttggta gctcagagaa
     1861 ccttcgaaaa accgccctgc aaggcggttt tttcgttttc agagcaagag attacgcgca
     1921 gaccaaaacg atctcaagaa gatcatctta ttaatcagat aaaatatttc taggatcatg
     1981 agcccgaagt ggcgagcccg atcttcccca tcggtgatgt cggcgatata ggcgccagca
     2041 accgcacctg tggcgccggt gatgccggcc acgatgcgtc cggcgtagag gatctgctca
     2101 tgtttgacag cttatcatcg atgcataatg tgcctgtcaa atggacgaag cagggattct
     2161 gcaaacccta tgctactccc acgagttgac agctagctca gtcctaggta taatactagt
     2221 gaagcacttc acagaagtgc atggagcaga aggtgatcga cagcaacaag tgagcggacg
     2281 caactagaag cacttcacag aagtgcatgg agcagaagcg cactccgtct ctgccctgtc
     2341 aaggcagctg ttttgcaagt tcgattatac ctttggggta aatggttgtc aatggcgggc
     2401 ccgcgacaga aaataggaag cctttctctg cacattcaac aaacaacatt tacagaatgc
     2461 cccaattatg ttatactaat gttgccttgc aataatgtga acgtgaaatg gagggatgtc
     2521 tgtgggattc aagaatcccg aagtgaagaa tcttgccgtc cgtacatgga cttgcccgaa
     2581 ctgtggggaa acccatgacc gagacgagaa cgctgcgctg aacattcggc gtgaagcgtt
     2641 ggtgggaggt ctgtccccac cttaaacgct catggaggct atgtcagacc tgcttcggcg
     2701 ggcaatggtc tgcgaagtga gaaccatgca tttctgtgaa ctgcttccaa aacgcactgc
     2761 ttcattataa tcctccctgt gcagttctgc cgaagcactt cacagaagtg catggagcag
     2821 aaggtgatcg acagcaacaa gtgagcggac gcaactagaa gcacttcaca gaagtgcatg
     2881 gagcagaaga agtcgtttat agcggtttgt ttacagggag ttagaagcac atttcaattt
     2941 ttcagtaaaa ctcctccagc tttgcagctg ggggagtttt ttactcccct cttgacaatt
     3001 atcttctacg tctgtagaat ataatcaaac tacaaccgta gaagatattc acagaaggga
     3061 ggataacgct atggaagctt gggcccgaac aaaaactcat ctcagaagag gatctgaata
     3121 gcgccgtcga ccatcatcat catcatcatt gagtttaaac ggtctacagc ttggctgttt
     3181 tggcggatga gagaagattt tcagcctgat acagattaaa tcagaacgca gaagcggtct
     3241 gataaaacag aatttgcctg gcggcagtag cgcggtggtc ccacctgacc ccatgccgaa
     3301 ctcagaagtg aaacgccgta gcgccgatgg tagtgtgggg tctacccatg cgagagtagg
     3361 gaactgccag gcatcaaata aaacgaaagg ctcagtcgaa agactgggcc tttcgtttta
     3421 tctgttgttt gtcggtgaac tgaattctca ctgcccgctt tccagtcggg aaacctgtcg
     3481 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     3541 cagggtggtt tttcttttca ccagtgagac tggcaacagc tgattgccct tcaccgcctg
     3601 gccctgagag agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg
     3661 tttgatggtg gttaacggcg ggatataaca tgagctatct tcggtatcgt cgtatcccac
     3721 taccgagata tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag
     3781 cgccatctga tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg
     3841 catggtttgt tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg
     3901 aatttgattg cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga
     3961 acttaatggg cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac
     4021 gcccagtcgc gtaccgtcct catgggagaa aataatactg ttgatgggtg tctggtcaga
     4081 gacatcaaga aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg
     4141 gtcatccagc ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac
     4201 cgccgcttta caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc
     4261 cagttgatcg gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag
     4321 actggaggtg gcaactccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg
     4381 gttgggaatg taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga
     4441 aacgtggctg gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc
     4501 tgcgacatcg tataacgtta ctggtttcat attcaccacc ctgaattgac tctcttccgg
     4561 gcgctatcat gccataccgc gaaaggtttt gcgccattcg atggcgcgcc gcttcgtcag
     4621 gccacatagc tttcttgttc tgatcggaac gatcgttggc tgtgttgaca attaatcatc
     4681 ggctcgtata atgtgtggaa ttgtgagcgc tcacaattag ctgtcaccgg atgtgctttc
     4741 cggtctgatg agtccgtgag gacgaaacag cctctacaaa taattttgtt taaagatcta
     4801 aaataaggag gaaaaaaaaa tgataaggaa taaggctttc gtggtcaggc tgtacccaaa
     4861 tgcggctcag actgaactga ttaaccgcac gctgggtagc gcaaggttcg tctacaacca
     4921 cttccttgcc cgtcgcattg cggcctacaa ggaaagcggg aagggactga cctacgggca
     4981 aacgagtagc gaactgaccc ttctgaagca ggctgaagaa acctcctggc tctcggaagt
     5041 agataagttt gctttgcaga actcgctgaa aaaccttgag accgcgtaca agaacttctt
     5101 tcggactgtg aagcagtccg gtaaaaaggt aggattccca cgtttcagaa agaagcgcac
     5161 gggagagtcc taccggactc aattcaccaa caacaacatc caaattgggg aaggtaggct
     5221 caaacttcct aagctgggat gggtgaaaac caagggccag caggatattc aagggaagat
     5281 tctgaatgtc actgtgcgcc gtattcacga aggccattac gaagcgtccg ttctctgtga
     5341 agtcgagatt ccctacctgc ctgcggctcc caagtttgca gcgggtgtgg atgtcggcat
     5401 caaggatttt gccatcgtga ccgatggcgt gaggtttaag catgaacaga atccgaaata
     5461 ttaccgctcc accctgaaaa gacttcgtaa agctcagcaa accctgtcca gacggaagaa
     5521 gggcagcgca cgttacggga aagcgaaaac caagctggct cggattcaca agcgcattgt
     5581 caataagcgt caggatttcc ttcacaagct caccacctcc ctggtgcgtg agtacgaaat
     5641 catcggaacc gaacacctta aacccgacaa catgcggaaa aatcgccgcc ttgcactgag
     5701 catcagtgat gcgggctggg gtgagttcat ccggcagttg gaatacaagg cagcgtggta
     5761 cgggcgactg gtatctaaag tcagccccta ctttccatct agccagttgt gtcatgactg
     5821 cggattcaag aatcccgaag tgaagaatct tgccgtccgt acatggactt gcccgaactg
     5881 tggggaaacc catgaccgag acgagaacgc tgcgctgaac attcggcgtg aagcgttggt
     5941 ggctgcggga atctcagaca ccttaaacgc tcatggaggc tatgtcagac ctgcttcggc
     6001 gggcaatggt ctgcgaagtg agaatcacgc gactttagtc gtgtgactcg aggagctcaa
     6061 ataaagccct gagtttaacc gctcggggct ttttgcgttt ctgagtagga caaatccgcc
     6121 gggagcggat ttgaacgttg cgaagcaacg gcccggaggg tggcgggcag gacgcccgcc
     6181 ataaactgcc aggcatca
//
