LOCUS       sequence-227894-        4816 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 4816)
  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 4816)
  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: pTW327
FEATURES             Location/Qualifiers
     source          1..4816
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     89..106
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(265..292)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(384..470)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     519..541
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     533..550
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     534..550
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     569..589
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    603..834
                     /gene="mutant version of attP"
                     /label=attP1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             complement(1230..1535)
                     /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(1332..1351)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1855..2538)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2453..2472
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2539..2641)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2786..3017)
                     /gene="mutant version of attP"
                     /label=attP2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     primer_bind     complement(3054..3073)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(3055..3073)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(3078..3094)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3078..3094)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3196..3215)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             3207..4016
                     /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(3274..3293)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      4163..4751
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4652..4671
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt gagctgatac
       61 cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag cggaggagcg
      121 cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt cattaatgca gctggcacga
      181 caggtttccc gactggaaag cgggcagtga gcgcaacgca attaatacgc gtaccgctag
      241 ccaggaagag tttgtagaaa cgcaaaaagg ccatccgtca ggatggcctt ctgcttagtt
      301 tgatgcctgg cagtttatgg cgggcgtcct gcccgccacc ctccgggccg ttgcttcaca
      361 acgttcaaat ccgctcccgg cggatttgtc ctactcagga gagcgttcac cgacaaacaa
      421 cagataaaac gaaaggccca gtcttccgac tgagcctttc gttttatttg atgcctggca
      481 gttccctact ctcgcgttaa cgctagcatg gatgttttcc cagtcacgac gttgtaaaac
      541 gacggccagt cttaagctcg gctcttcacg caaatgggcg gtaggcgtgg actggagacc
      601 ccaaataatg attttatttt gactgatagt gacctgttcg ttgcaacaaa ttgatgagca
      661 atgctttttt ataatgccaa ctttgtacaa aaaagctgaa cgagaaacgt aaaatgatat
      721 aaatatcaat atattaaatt agattttgca taaaaaacag actacataat actgtaaaac
      781 acaacatatc cagtcactat gaatcaacta cttagatggt attagtgacc tgtagtcgac
      841 cgacagcctt ccaaatgttc ttcgggtgat gctgccaact tagtcgaccg acagccttcc
      901 aaatgttctt ctcaaacgga atcgtcgtat ccagcctact cgctattgtc ctcaatgccg
      961 tattaaatca taaaaagaaa taagaaaaag aggtgcgagc ctcttttttg tgtgacaaaa
     1021 taaaaacatc tacctattca tatacgctag tgtcatagtc ctgaaaatca tctgcatcaa
     1081 gaacaatttc acaactctta tacttttctc ttacaagtcg ttcggcttca tctggatttt
     1141 cagcctctat acttactaaa cgtgataaag tttctgtaat ttctactgta tcgacctgca
     1201 gactggctgt gtataaggga gcctgacatt tatattcccc agaacatcag gttaatggcg
     1261 tttttgatgt cattttcgcg gtggctgaga tcagccactt cttccccgat aacggagacc
     1321 ggcacactgg ccatatcggt ggtcatcatg cgccagcttt catccccgat atgcaccacc
     1381 gggtaaagtt cacgggagac tttatctgac agcagacgtg cactggccag ggggatcacc
     1441 atccgtcgcc cgggcgtgtc aataatatca ctctgtacat ccacaaacag acgataacgg
     1501 ctctctcttt tataggtgta aaccttaaac tgcatttcac cagcccctgt tctcgtcagc
     1561 aaaagagccg ttcatttcaa taaaccgggc gacctcagcc atcccttcct gattttccgc
     1621 tttccagcgt tcggcacgca gacgacgggc ttcattctgc atggttgtgc ttaccagacc
     1681 ggagatattg acatcatata tgccttgagc aactgatagc tgtcgctgtc aactgtcact
     1741 gtaatacgct gcttcatagc atacctcttt ttgacatact tcgggtatac atatcagtat
     1801 atattcttat accgcaaaaa tcagcgcgca aatacgcata ctgttatctg gcttttagta
     1861 agccggatcc acgcggcgtt tacgcccccc ctgccactca tcgcagtact gttgtaattc
     1921 attaagcatt ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag
     1981 cggcatcagc accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa
     2041 gaagttgtcc atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc
     2101 tgagacgaaa aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta
     2161 acacgccaca tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact
     2221 ccagagcgat gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact
     2281 atcccatatc accagctcac cgtctttcat tgccatacgg aattccggat gagcattcat
     2341 caggcgggca agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt
     2401 ctttaaaaag gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga
     2461 ctgaaatgcc tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc
     2521 agtgattttt ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa
     2581 tacgcccggt agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc
     2641 aacgtctcat tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg
     2701 atttatttat tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc
     2761 gggtgatgct gccaacttag tcgactacag gtcactaata ccatctaagt agttgattca
     2821 tagtgactgg atatgttgtg ttttacagta ttatgtagtc tgttttttat gcaaaatcta
     2881 atttaatata ttgatattta tatcatttta cgtttctcgt tcagctttct tgtacaaagt
     2941 tggcattata agaaagcatt gcttatcaat ttgttgcaac gaacaggtca ctatcagtca
     3001 aaataaaatc attatttgcc atccagctga tggtctcctt cacgaagagc atcccctata
     3061 gtgagtcgta ttacatggtc atagctgttt cctggcagct ctggcccgtg tctcaaaatc
     3121 tctgatgtta cattgcacaa gataaaaata tatcatcatg aacaataaaa ctgtctgctt
     3181 acataaacag taatacaagg ggtgttatga gccatattca acgggaaacg tcgaggccgc
     3241 gattaaattc caacatggat gctgatttat atgggtataa atgggctcgc gataatgtcg
     3301 ggcaatcagg tgcgacaatc tatcgcttgt atgggaagcc cgatgcgcca gagttgtttc
     3361 tgaaacatgg caaaggtagc gttgccaatg atgttacaga tgagatggtc agactaaact
     3421 ggctgacgga atttatgcct cttccgacca tcaagcattt tatccgtact cctgatgatg
     3481 catggttact caccactgcg atccccggaa aaacagcatt ccaggtatta gaagaatatc
     3541 ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt cctgcgccgg ttgcattcga
     3601 ttcctgtttg taattgtcct tttaacagcg atcgcgtatt tcgtctcgct caggcgcaat
     3661 cacgaatgaa taacggtttg gttgatgcga gtgattttga tgacgagcgt aatggctggc
     3721 ctgttgaaca agtctggaaa gaaatgcata aacttttgcc attctcaccg gattcagtcg
     3781 tcactcatgg tgatttctca cttgataacc ttatttttga cgaggggaaa ttaataggtt
     3841 gtattgatgt tggacgagtc ggaatcgcag accgatacca ggatcttgcc atcctatgga
     3901 actgcctcgg tgagttttct ccttcattac agaaacggct ttttcaaaaa tatggtattg
     3961 ataatcctga tatgaataaa ttgcagtttc atttgatgct cgatgagttt ttctaatcag
     4021 aattggttaa ttggttgtaa cactggcaga gcattacgct gacttgacgg gacggcgcaa
     4081 gctcatgacc aaaatccctt aacgtgagtt acgcgtcgtt ccactgagcg tcagaccccg
     4141 tagaaaagat caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc
     4201 aaacaaaaaa accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc
     4261 tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt
     4321 agccgtagtt aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc
     4381 taatcctgtt accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact
     4441 caagacgata gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac
     4501 agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag
     4561 aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg
     4621 gaacaggaga gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg
     4681 tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga
     4741 gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt
     4801 ttgctcacat gttttc
//
