LOCUS       sequence-274122-        5364 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 5364)
  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 5364)
  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..5364
                     /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..1157
                     /codon_start=1
                     /gene="cat"
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGA"
     primer_bind     complement(564..583)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1499..1804
                     /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     1683..1702
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2194..2317)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     3'UTR           2342..2467
                     /label=Xenopus globin 3'-UTR
                     /note="3'-UTR of the major beta-globin gene of Xenopus 
                     laevis"
     primer_bind     complement(2553..2572)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2554..2572)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2579..2596)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2579..2595)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2588..2610)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      complement(2736..3191)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2873..2894)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3085..3104
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3218..3322
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3323..3391,3392..4183)
                     /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(3541..3560)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4354..4942
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4843..4862
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5096..5113
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5230..5251
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5266..5271,5272..5289,5290..5296)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5304..5320
                     /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     5309..5331
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5328..5344
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5328..5344
                     /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 tctgtgatgg cttccatgtc ggcagaatgc ttaatgaatt acaacagtac tgcgatgagt
     1141 ggcagggcgg ggcgtaatcg cgtggatccg gcttactaaa agccagataa cagtatgcgt
     1201 atttgcgcgc tgatttttgc ggtataagaa tatatactga tatgtatacc cgaagtatgt
     1261 caaaaagagg tgtgctatga agcagcgtat tacagtgaca gttgacagcg acagctatca
     1321 gttgctcaag gcatatatga tgtcaatatc tccggtctgg taagcacaac catgcagaat
     1381 gaagcccgtc gtctgcgtgc cgaacgctgg aaagcggaaa atcaggaagg gatggctgag
     1441 gtcgcccggt ttattgaaat gaacggctct tttgctgacg agaacaggga ctggtgaaat
     1501 gcagtttaag gtttacacct ataaaagaga gagccgttat cgtctgtttg tggatgtaca
     1561 gagtgatatt attgacacgc ccgggcgacg gatggtgatc cccctggcca gtgcacgtct
     1621 gctgtcagat aaagtctccc gtgaacttta cccggtggtg catatcgggg atgaaagctg
     1681 gcgcatgatg accaccgata tggccagtgt gccggtctcc gttatcgggg aagaagtggc
     1741 tgatctcagc caccgcgaaa atgacatcaa aaacgccatt aacctgatgt tctggggaat
     1801 ataaatgtca ggctccctta tacacagcca gtctgcaggt cgatacagta gaaattacag
     1861 aaactttatc acgtttagta agtatagagg ctgaaaatcc agatgaagcc gaacgacttg
     1921 taagagaaaa gtataagagt tgtgaaattg ttcttgatgc agatgatttt caggactatg
     1981 acactagcgt atatgaatag gtagatgttt ttattttgtc acacaaaaaa gaggctcgca
     2041 cctctttttc ttatttcttt ttatgattta atacggcatt gaggacaata gcgagtaggc
     2101 tggatacgac gattccgttt gagaagaaca tttggaaggc tgtcggtcga gctcgaattc
     2161 tacaggtcac taataccatc taagtagttg attcatagtg actgcatatg ttgtgtttta
     2221 cagtattatg tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca
     2281 ttttacgttt ctcgttcaac tttattatac atagttgctc gagcggatct ggttaccact
     2341 aaaccagcct caagaacacc cgaatggagt ctctaagcta cataatacca acttacactt
     2401 tacaaaatgt tgtcccccaa aatgtagcca ttcgtatctg ctcctaataa aaagaaagtt
     2461 tcttcacatt ctaaaaaaaa aaaaaaaaaa aaaaaaaaaa accccccccc ccccctgcag
     2521 ggccaagtcg gcctctagag gtacccaatt cgccctatag tgagtcgtat tacaattcac
     2581 tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa cttaatcgcc
     2641 ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc accgatcgcc
     2701 cttcccaaca gttgcgcagc ctgaatggcg aatggaaatt gtaagcgtta atattttgtt
     2761 aaaattcgcg ttaaattttt gttaaatcag ctcatttttt aaccaatagg ccgaaatcgg
     2821 caaaatccct tataaatcaa aagaatagac cgagataggg ttgagtgttg ttccagtttg
     2881 gaacaagagt ccactattaa agaacgtgga ctccaacgtc aaagggcgaa aaaccgtcta
     2941 tcagggcgat ggcccactac gtgaaccatc accctaatca agttttttgg ggtcgaggtg
     3001 ccgtaaagca ctaaatcgga accctaaagg gagcccccga tttagagctt gacggggaaa
     3061 gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa ggagcgggcg ctagggcgct
     3121 ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc gccgcgctta atgcgccgct
     3181 acagggcgcg tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt
     3241 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca
     3301 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt
     3361 ttttgcggca ttttgccttc ctgtttttgc tcacccagaa acgctggtga aagtaaaaga
     3421 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa
     3481 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct
     3541 gctatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat
     3601 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcttacgga
     3661 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc
     3721 caacttactt ctgacaacga tcggaggacc gaaggagcta accgcttttt tgcacaacat
     3781 gggggatcat gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa
     3841 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aactattaac
     3901 tggcgaacta cttactctag cttcccggca acaattaata gactggatgg aggcggataa
     3961 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttattg ctgataaatc
     4021 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc
     4081 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggatg aacgaaatag
     4141 acagatcgct gagataggtg cctcactgat taagcattgg taactgtcag accaagttta
     4201 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa
     4261 gatccttttt gataatctca tgaccaaaat cccttaacgt gagttttcgt tccactgagc
     4321 gtcagacccc gtagaaaaga tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat
     4381 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga
     4441 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt
     4501 ccttctagtg tagccgtagt taggccacca cttcaagaac tctgtagcac cgcctacata
     4561 cctcgctctg ctaatcctgt taccagtggc tgctgccagt ggcgataagt cgtgtcttac
     4621 cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg
     4681 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagat acctacagcg
     4741 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggtaag
     4801 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct
     4861 ttatagtcct gtcgggtttc gccacctctg acttgagcgt cgatttttgt gatgctcgtc
     4921 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt
     4981 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgattctg tggataaccg
     5041 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga
     5101 gtcagtgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg
     5161 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg
     5221 caacgcaatt aatgtgagtt agctcactca ttaggcaccc caggctttac actttatgct
     5281 tccggctcgt atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta
     5341 tgaccatgat tacgccaagc tcga
//
