LOCUS       sequence-244252-        4660 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 4660)
  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 4660)
  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: pTW063
FEATURES             Location/Qualifiers
     source          1..4660
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(146..469)
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     misc_feature    396..452
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(465..481)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(465..481)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(478..500)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    489..505
                     /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)."
     promoter        complement(join(513..519,520..537,538..543))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    558..579
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     CDS             complement(1098..1403)
                     /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(1200..1219)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1723..2406)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2321..2340
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2407..2509)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2670..2687)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2841..3429)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2921..2940)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3600..4391,4392..4460))
                     /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     4223..4242
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4461..4565)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4633..4651
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgagctcggt acccggggat
      421 cctctagagt cgacctgcag gcatgcaagc ttggcgtaat catggtcata gctgtttcct
      481 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
      541 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
      601 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
      661 agaggcggtt tgcgtattgg gcgctcttcc gctggtctcc gactgtcgac cgacagcctt
      721 ccaaatgttc ttctccaacg gaatcggcgt gtccaccgac agccttccaa atgttcttct
      781 caaacggaat cgtcgtatcc agcctactcg ctattgtcct caatgccgta ttaaatcata
      841 aaaagaaata agaaaaagag gtgcgagcct cttttttgtg tgacaaaata aaaacatcta
      901 cctattcata tacgctagtg tcatagtcct gaaaatcatc tgcatcaaga acaatttcac
      961 aactcttata cttttctctt acaagtcgtt cggcttcatc tggattttca gcctctatac
     1021 ttactaaacg tgataaagtt tctgtaattt ctactgtatc gacctgcaga ctggctgtgt
     1081 ataagggagc ctgacattta tattccccag aacatcaggt taatggcgtt tttgatgtca
     1141 ttttcgcggt ggctgagatc agccacttct tccccgataa cggagaccgg cacactggcc
     1201 atatcggtgg tcatcatgcg ccagctttca tccccgatat gcaccaccgg gtaaagttca
     1261 cgggagactt tatctgacag cagacgtgca ctggccaggg ggatcaccat ccgtcgcccg
     1321 ggcgtgtcaa taatatcact ctgtacatcc acaaacagac gataacggct ctctctttta
     1381 taggtgtaaa ccttaaactg catttcacca gcccctgttc tcgtcagcaa aagagccgtt
     1441 catttcaata aaccgggcga cctcagccat cccttcctga ttttccgctt tccagcgttc
     1501 ggcacgcaga cgacgggctt cattctgcat ggttgtgctt accagaccgg agatattgac
     1561 atcatatatg ccttgagcaa ctgatagctg tcgctgtcaa ctgtcactgt aatacgctgc
     1621 ttcatagcat acctcttttt gacatacttc gggtatacat atcagtatat attcttatac
     1681 cgcaaaaatc agcgcgcaaa tacgcatact gttatctggc ttttagtaag ccggatccac
     1741 gcggcgttta cgccccccct gccactcatc gcagtactgt tgtaattcat taagcattct
     1801 gccgacatgg aagccatcac aaacggcatg atgaacctga atcgccagcg gcatcagcac
     1861 cttgtcgcct tgcgtataat atttgcccat ggtgaaaacg ggggcgaaga agttgtccat
     1921 attggccacg tttaaatcaa aactggtgaa actcacccag ggattggctg agacgaaaaa
     1981 catattctca ataaaccctt tagggaaata ggccaggttt tcaccgtaac acgccacatc
     2041 ttgcgaatat atgtgtagaa actgccggaa atcgtcgtgg tattcactcc agagcgatga
     2101 aaacgtttca gtttgctcat ggaaaacggt gtaacaaggg tgaacactat cccatatcac
     2161 cagctcaccg tctttcattg ccatacggaa ttccggatga gcattcatca ggcgggcaag
     2221 aatgtgaata aaggccggat aaaacttgtg cttatttttc tttacggtct ttaaaaaggc
     2281 cgtaatatcc agctgaacgg tctggttata ggtacattga gcaactgact gaaatgcctc
     2341 aaaatgttct ttacgatgcc attgggatat atcaacggtg gtatatccag tgattttttt
     2401 ctccatttta gcttccttag ctcctgaaaa tctcgataac tcaaaaaata cgcccggtag
     2461 tgatcttatt tcattatggt gaaagttgga acctcttacg tgccgatcaa cgtctcattt
     2521 tcgccaaaag ttggcccagg gcttcccggt atcaacaggg acaccaggat ttatttattc
     2581 tgcgaagtga tcttccgtca caggtattta ttcggcgcaa agtgcgtcgg gtgatgctgc
     2641 caacttagtc gaccacctga gaccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     2701 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     2761 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     2821 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     2881 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     2941 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     3001 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     3061 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     3121 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     3181 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     3241 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     3301 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     3361 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     3421 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     3481 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     3541 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     3601 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     3661 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     3721 agtgctgcaa tgataccgcg agatccacgc tcaccggctc cagatttatc agcaataaac
     3781 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     3841 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     3901 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     3961 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     4021 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     4081 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     4141 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     4201 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     4261 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     4321 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     4381 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     4441 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     4501 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     4561 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
     4621 ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
