LOCUS       sequence-227887-        4794 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Destination Vector.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4794)
  AUTHORS   Wagner TE, Becraft JR, Bodner K, Teague B, Zhang X, Woo A, Porter E,
            Alburquerque B, Dobosh B, Andries O, Sanders NN, Beal J, Densmore D,
            Kitada T, Weiss R
  TITLE     Small-molecule-based regulation of RNA-delivered circuits in 
            mammalian cells.
  JOURNAL   Nat Chem Biol. 2018 Nov;14(11):1043-1050. doi: 
            10.1038/s41589-018-0146-9. Epub 2018 Oct 16.
  PUBMED    30327560
REFERENCE   2  (bases 1 to 4794)
  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: 
            "10.1038/s41589-018-0146-9"; journalName: "Nat Chem Biol"; date: 
            "2018-11"; volume: "14"; issue: "11"; pages: "1043-1050"
COMMENT     Sequence Label: pTW324
FEATURES             Location/Qualifiers
     source          1..4794
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     92..109
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(268..295)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(387..473)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     522..544
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     536..553
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     537..553
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    584..815
                     /gene="mutant version of attP"
                     /label=attP1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(1211..1516)
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="MQFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDK
                     VSRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     complement(1313..1332)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1836..2519)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2434..2453
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2520..2622)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2767..2998)
                     /gene="mutant version of attP"
                     /label=attP2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3035..3054)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3036..3054)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3059..3075)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3059..3075)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3177..3196)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3188..3997
                     /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="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(3255..3274)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4144..4732
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4633..4652
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg agtgagctga
       61 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggagga
      121 gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca
      181 cgacaggttt cccgactgga aagcgggcag tgagcgcaac gcaattaata cgcgtaccgc
      241 tagccaggaa gagtttgtag aaacgcaaaa aggccatccg tcaggatggc cttctgctta
      301 gtttgatgcc tggcagttta tggcgggcgt cctgcccgcc accctccggg ccgttgcttc
      361 acaacgttca aatccgctcc cggcggattt gtcctactca ggagagcgtt caccgacaaa
      421 caacagataa aacgaaaggc ccagtcttcc gactgagcct ttcgttttat ttgatgcctg
      481 gcagttccct actctcgcgt taacgctagc atggatgttt tcccagtcac gacgttgtaa
      541 aacgacggcc agtcttaagc tcggctcttc gactggagac cccaaataat gattttattt
      601 tgactgatag tgacctgttc gttgcaacaa attgatgagc aatgcttttt tataatgcca
      661 actttgtaca aaaaagctga acgagaaacg taaaatgata taaatatcaa tatattaaat
      721 tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat ccagtcacta
      781 tgaatcaact acttagatgg tattagtgac ctgtagtcga ccgacagcct tccaaatgtt
      841 cttcgggtga tgctgccaac ttagtcgacc gacagccttc caaatgttct tctcaaacgg
      901 aatcgtcgta tccagcctac tcgctattgt cctcaatgcc gtattaaatc ataaaaagaa
      961 ataagaaaaa gaggtgcgag cctctttttt gtgtgacaaa ataaaaacat ctacctattc
     1021 atatacgcta gtgtcatagt cctgaaaatc atctgcatca agaacaattt cacaactctt
     1081 atacttttct cttacaagtc gttcggcttc atctggattt tcagcctcta tacttactaa
     1141 acgtgataaa gtttctgtaa tttctactgt atcgacctgc agactggctg tgtataaggg
     1201 agcctgacat ttatattccc cagaacatca ggttaatggc gtttttgatg tcattttcgc
     1261 ggtggctgag atcagccact tcttccccga taacggagac cggcacactg gccatatcgg
     1321 tggtcatcat gcgccagctt tcatccccga tatgcaccac cgggtaaagt tcacgggaga
     1381 ctttatctga cagcagacgt gcactggcca gggggatcac catccgtcgc ccgggcgtgt
     1441 caataatatc actctgtaca tccacaaaca gacgataacg gctctctctt ttataggtgt
     1501 aaaccttaaa ctgcatttca ccagcccctg ttctcgtcag caaaagagcc gttcatttca
     1561 ataaaccggg cgacctcagc catcccttcc tgattttccg ctttccagcg ttcggcacgc
     1621 agacgacggg cttcattctg catggttgtg cttaccagac cggagatatt gacatcatat
     1681 atgccttgag caactgatag ctgtcgctgt caactgtcac tgtaatacgc tgcttcatag
     1741 catacctctt tttgacatac ttcgggtata catatcagta tatattctta taccgcaaaa
     1801 atcagcgcgc aaatacgcat actgttatct ggcttttagt aagccggatc cacgcggcgt
     1861 ttacgccccc cctgccactc atcgcagtac tgttgtaatt cattaagcat tctgccgaca
     1921 tggaagccat cacaaacggc atgatgaacc tgaatcgcca gcggcatcag caccttgtcg
     1981 ccttgcgtat aatatttgcc catggtgaaa acgggggcga agaagttgtc catattggcc
     2041 acgtttaaat caaaactggt gaaactcacc cagggattgg ctgagacgaa aaacatattc
     2101 tcaataaacc ctttagggaa ataggccagg ttttcaccgt aacacgccac atcttgcgaa
     2161 tatatgtgta gaaactgccg gaaatcgtcg tggtattcac tccagagcga tgaaaacgtt
     2221 tcagtttgct catggaaaac ggtgtaacaa gggtgaacac tatcccatat caccagctca
     2281 ccgtctttca ttgccatacg gaattccgga tgagcattca tcaggcgggc aagaatgtga
     2341 ataaaggccg gataaaactt gtgcttattt ttctttacgg tctttaaaaa ggccgtaata
     2401 tccagctgaa cggtctggtt ataggtacat tgagcaactg actgaaatgc ctcaaaatgt
     2461 tctttacgat gccattggga tatatcaacg gtggtatatc cagtgatttt tttctccatt
     2521 ttagcttcct tagctcctga aaatctcgat aactcaaaaa atacgcccgg tagtgatctt
     2581 atttcattat ggtgaaagtt ggaacctctt acgtgccgat caacgtctca ttttcgccaa
     2641 aagttggccc agggcttccc ggtatcaaca gggacaccag gatttattta ttctgcgaag
     2701 tgatcttccg tcacaggtat ttattcggcg caaagtgcgt cgggtgatgc tgccaactta
     2761 gtcgactaca ggtcactaat accatctaag tagttgattc atagtgactg gatatgttgt
     2821 gttttacagt attatgtagt ctgtttttta tgcaaaatct aatttaatat attgatattt
     2881 atatcatttt acgtttctcg ttcagctttc ttgtacaaag ttggcattat aagaaagcat
     2941 tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgc
     3001 catccagctg atggtctcct tcacgaagag catcccctat agtgagtcgt attacatggt
     3061 catagctgtt tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt acattgcaca
     3121 agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag
     3181 gggtgttatg agccatattc aacgggaaac gtcgaggccg cgattaaatt ccaacatgga
     3241 tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat
     3301 ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag
     3361 cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc
     3421 tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc
     3481 gatccccgga aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
     3541 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
     3601 ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt
     3661 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
     3721 agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
     3781 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
     3841 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
     3901 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
     3961 attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta
     4021 acactggcag agcattacgc tgacttgacg ggacggcgca agctcatgac caaaatccct
     4081 taacgtgagt tacgcgtcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     4141 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     4201 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     4261 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     4321 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     4381 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     4441 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     4501 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     4561 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     4621 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     4681 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     4741 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgtt
//
