LOCUS       sequence-325299-        5368 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  166571: Multisite gateway destination vector for in vitro 
            transcription of RNA using T3. Accepts one pME and one p3E entry 
            clone..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5368)
  AUTHORS   Hall TE, Martel N, Ariotti N, Xiong Z, Lo HP, Ferguson C, Rae J, Lim
            YW, Parton RG
  TITLE     In vivo cell biological screening identifies an endocytic capture 
            mechanism for T-tubule formation.
  JOURNAL   Nat Commun. 2020 Jul 24;11(1):3711. doi: 10.1038/s41467-020-17486-w.
  PUBMED    32709891
REFERENCE   2  (bases 1 to 5368)
  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: "Nat 
            Commun."; date: "2020-07-24"; volume: "11(1):3711. doi"; pages: " 
            10.1038/s41467-020-17486-w"
COMMENT     Sequence Label: pT3TS-Dest_R1-R3
FEATURES             Location/Qualifiers
     source          1..5368
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..19
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     5'UTR           44..86
                     /label=Xenopus globin 5'-UTR
                     /note="translational enhancer from the 5'-UTR of the major 
                     beta-globin gene of Xenopus laevis"
     protein_bind    94..217
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        395..497
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             498..1181
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(564..583)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1501..1806
                     /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     1685..1704
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2196..2319)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     3'UTR           2344..2469
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     primer_bind     complement(2556..2575)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2557..2575)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2582..2599)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2582..2598)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2591..2613)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2740..3195)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2877..2898)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3089..3108
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3222..3326
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3327..3395,3396..4187)
                     /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     complement(3545..3564)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4358..4946
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4847..4866
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5100..5117
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5234..5255
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5270..5275,5276..5293,5294..5300)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5308..5324
                     /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)."
     primer_bind     5313..5335
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5332..5348
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5332..5348
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
ORIGIN
        1 aattaaccct cactaaaggg aacaaaagct ggagctaagc ttgcttgttc tttttgcaga
       61 agctcagaat aaacgctcaa ctttggcaga tctcaagttt gtacaaaaaa gctgaacgag
      121 aaacgtaaaa tgatataaat atcaatatat taaattagat tttgcataaa aaacagacta
      181 cataatactg taaaacacaa catatccagt cactatgaat caactactta gatggtatta
      241 gtgacctgta gtcgactaag ttggcagcat cacccgacgc actttgcgcc gaataaatac
      301 ctgtgacgga agatcacttc gcagaataaa taaatcctgg tgtccctgtt gataccggga
      361 agccctgggc caacttttgg cgaaaatgag acgttgatcg gcacgtaaga ggttccaact
      421 ttcaccataa tgaaataaga tcactaccgg gcgtattttt tgagttatcg agattttcag
      481 gagctaagga agctaaaatg gagaaaaaaa tcactggata taccaccgtt gatatatccc
      541 aatggcatcg taaagaacat tttgaggcat ttcagtcagt tgctcaatgt acctataacc
      601 agaccgttca gctggatatt acggcctttt taaagaccgt aaagaaaaat aagcacaagt
      661 tttatccggc ctttattcac attcttgccc gcctgatgaa tgctcatccg gaattccgta
      721 tggcaatgaa agacggtgag ctggtgatat gggatagtgt tcacccttgt tacaccgttt
      781 tccatgagca aactgaaacg ttttcatcgc tctggagtga ataccacgac gatttccggc
      841 agtttctaca catatattcg caagatgtgg cgtgttacgg tgaaaacctg gcctatttcc
      901 ctaaagggtt tattgagaat atgtttttcg tctcagccaa tccctgggtg agtttcacca
      961 gttttgattt aaacgtggcc aatatggaca acttcttcgc ccccgttttc accatgggca
     1021 aatattatac gcaaggcgac aaggtgctga tgccgctggc gattcaggtt catcatgccg
     1081 tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt
     1141 ggcagggggg gcgtaaacgc cgcgtggatc cggcttacta aaagccagat aacagtatgc
     1201 gtatttgcgc gctgattttt gcggtataag aatatatact gatatgtata cccgaagtat
     1261 gtcaaaaaga ggtatgctat gaagcagcgt attacagtga cagttgacag cgacagctat
     1321 cagttgctca aggcatatat gatgtcaata tctccggtct ggtaagcaca accatgcaga
     1381 atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga aaatcaggaa gggatggctg
     1441 aggtcgcccg gtttattgaa atgaacggct cttttgctga cgagaacagg ggctggtgaa
     1501 atgcagttta aggtttacac ctataaaaga gagagccgtt atcgtctgtt tgtggatgta
     1561 cagagtgata ttattgacac gcccgggcga cggatggtga tccccctggc cagtgcacgt
     1621 ctgctgtcgg ataaagtctc ccgtgaactt tacccggtgg tgcatatcgg ggatgaaagc
     1681 tggcgcatga tgaccaccga tatggccagt gtgccggtct ccgttatcgg ggaagaagtg
     1741 gctgatctca gccaccgcga aaatgacatc aaaaacgcca ttaacctgat gttctgggga
     1801 atataaatgt caggctccct tatacacagc cagtctgcag gtcgatacag tagaaattac
     1861 agaaacttta tcacgtttag taagtataga ggctgaaaat ccagatgaag ccgaacgact
     1921 tgtaagagaa aagtataaga gttgtgaaat tgttcttgat gcagatgatt ttcaggacta
     1981 tgacactagc gtatatgaat aggtagatgt ttttattttg tcacacaaaa aagaggctcg
     2041 cacctctttt tcttatttct ttttatgatt taatacggca ttgaggacaa tagcgagtag
     2101 gctggatacg acgattccgt ttgagaagaa catttggaag gctgtcggtc gagctcgaat
     2161 tctacaggtc actaatacca tctaagtagt tgattcatag tgactgcata tgttgtgttt
     2221 tacagtatta tgtagtctgt tttttatgca aaatctaatt taatatattg atatttatat
     2281 cattttacgt ttctcgttca actttattat acatagttgc tcgagcggat ctggttacca
     2341 ctaaaccagc ctcaagaaca cccgaatgga gtctctaagc tacataatac caacttacac
     2401 tttacaaaat gttgtccccc aaaatgtagc cattcgtatc tgctcctaat aaaaagaaag
     2461 tttcttcaca ttctaaaaaa aaaaaaaaaa aaaaaaaaaa aaancccccc cccccccctg
     2521 cagggccaag tcggcctcta gaggtaccca attcgcccta tagtgagtcg tattacaatt
     2581 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     2641 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     2701 gcccttccca acagttgcgc agcctgaatg gcgaatggca aattgtaagc gttaatattt
     2761 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     2821 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     2881 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     2941 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     3001 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     3061 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     3121 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     3181 cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     3241 tatttttcta aatacattca aatatgtatc tgctcatgag acaataaccc tgataaatgc
     3301 ttcaataata ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc
     3361 ccttttttgc ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa
     3421 aagatgctga agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg
     3481 gtaagatcct tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag
     3541 ttctgctatg tggcgcggta ttatcccgta ttgacgccgg gcaagagcaa ctcggtcgcc
     3601 gcatacacta ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta
     3661 cggatggcat gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg
     3721 cggccaactt acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca
     3781 acatggggga tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac
     3841 caaacgacga gcgtgacacc acgatgcctg tagcaatggc aacaacgttg cgcaaactat
     3901 taactggcga actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg
     3961 ataaagttgc aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata
     4021 aatctggagc cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta
     4081 agccctcccg tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa
     4141 atagacagat cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag
     4201 tttactcata tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg
     4261 tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact
     4321 gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg
     4381 taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc
     4441 aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata
     4501 ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta
     4561 catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc
     4621 ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg
     4681 ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac
     4741 agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg
     4801 taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt
     4861 atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct
     4921 cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg
     4981 ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata
     5041 accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca
     5101 gcgagtcagt gagcgaggaa gcggaagagc gcccaatacg caaaccgcct ctccccgcgc
     5161 gttggccgat tcattaatgc agctggcacg acaggtttcc cgactggaaa gcgggcagtg
     5221 agcgcaacgc aattaatgtg agttagctca ctcattaggc accccaggct ttacacttta
     5281 tgcttccggc tcgtatgttg tgtggaattg tgagcggata acaatttcac acaggaaaca
     5341 gctatgacca tgattacgcc aagctcga
//
