LOCUS       sequence-244253-        4662 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 4662)
  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 4662)
  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: pTW065
FEATURES             Location/Qualifiers
     source          1..4662
                     /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(1100..1405)
                     /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(1202..1221)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1725..2408)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2323..2342
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2409..2511)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     primer_bind     complement(2672..2689)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2843..3431)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2923..2942)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3602..4393,4394..4462))
                     /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     4225..4244
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4463..4567)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     4635..4653
                     /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 ataagtcgac cgacagcctt
      721 ccaaatgttc ttttcaaacg ggatcgccat aagtcgaccg acagccttcc aaatgttctt
      781 ctcaaacgga atcgtcgtat ccagcctact cgctattgtc ctcaatgccg tattaaatca
      841 taaaaagaaa taagaaaaag aggtgcgagc ctcttttttg tgtgacaaaa taaaaacatc
      901 tacctattca tatacgctag tgtcatagtc ctgaaaatca tctgcatcaa gaacaatttc
      961 acaactctta tacttttctc ttacaagtcg ttcggcttca tctggatttt cagcctctat
     1021 acttactaaa cgtgataaag tttctgtaat ttctactgta tcgacctgca gactggctgt
     1081 gtataaggga gcctgacatt tatattcccc agaacatcag gttaatggcg tttttgatgt
     1141 cattttcgcg gtggctgaga tcagccactt cttccccgat aacggagacc ggcacactgg
     1201 ccatatcggt ggtcatcatg cgccagcttt catccccgat atgcaccacc gggtaaagtt
     1261 cacgggagac tttatctgac agcagacgtg cactggccag ggggatcacc atccgtcgcc
     1321 cgggcgtgtc aataatatca ctctgtacat ccacaaacag acgataacgg ctctctcttt
     1381 tataggtgta aaccttaaac tgcatttcac cagcccctgt tctcgtcagc aaaagagccg
     1441 ttcatttcaa taaaccgggc gacctcagcc atcccttcct gattttccgc tttccagcgt
     1501 tcggcacgca gacgacgggc ttcattctgc atggttgtgc ttaccagacc ggagatattg
     1561 acatcatata tgccttgagc aactgatagc tgtcgctgtc aactgtcact gtaatacgct
     1621 gcttcatagc atacctcttt ttgacatact tcgggtatac atatcagtat atattcttat
     1681 accgcaaaaa tcagcgcgca aatacgcata ctgttatctg gcttttagta agccggatcc
     1741 acgcggcgtt tacgcccccc ctgccactca tcgcagtact gttgtaattc attaagcatt
     1801 ctgccgacat ggaagccatc acaaacggca tgatgaacct gaatcgccag cggcatcagc
     1861 accttgtcgc cttgcgtata atatttgccc atggtgaaaa cgggggcgaa gaagttgtcc
     1921 atattggcca cgtttaaatc aaaactggtg aaactcaccc agggattggc tgagacgaaa
     1981 aacatattct caataaaccc tttagggaaa taggccaggt tttcaccgta acacgccaca
     2041 tcttgcgaat atatgtgtag aaactgccgg aaatcgtcgt ggtattcact ccagagcgat
     2101 gaaaacgttt cagtttgctc atggaaaacg gtgtaacaag ggtgaacact atcccatatc
     2161 accagctcac cgtctttcat tgccatacgg aattccggat gagcattcat caggcgggca
     2221 agaatgtgaa taaaggccgg ataaaacttg tgcttatttt tctttacggt ctttaaaaag
     2281 gccgtaatat ccagctgaac ggtctggtta taggtacatt gagcaactga ctgaaatgcc
     2341 tcaaaatgtt ctttacgatg ccattgggat atatcaacgg tggtatatcc agtgattttt
     2401 ttctccattt tagcttcctt agctcctgaa aatctcgata actcaaaaaa tacgcccggt
     2461 agtgatctta tttcattatg gtgaaagttg gaacctctta cgtgccgatc aacgtctcat
     2521 tttcgccaaa agttggccca gggcttcccg gtatcaacag ggacaccagg atttatttat
     2581 tctgcgaagt gatcttccgt cacaggtatt tattcggcgc aaagtgcgtc gggtgatgct
     2641 gccaacttag tcgacttcat gagaccgctt cctcgctcac tgactcgctg cgctcggtcg
     2701 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     2761 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     2821 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     2881 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     2941 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     3001 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     3061 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     3121 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     3181 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     3241 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     3301 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     3361 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     3421 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     3481 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     3541 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     3601 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     3661 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     3721 ccagtgctgc aatgataccg cgagatccac gctcaccggc tccagattta tcagcaataa
     3781 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     3841 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     3901 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     3961 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     4021 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     4081 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     4141 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     4201 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     4261 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     4321 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     4381 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     4441 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     4501 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     4561 ttccgcgcac atttccccga aaagtgccac ctgacgtcta agaaaccatt attatcatga
     4621 cattaaccta taaaaatagg cgtatcacga ggccctttcg tc
//
