LOCUS       sequence-341989-        5547 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite gateway destination vector for cell free expression (takes
            two cassettes). Parton lab clone JBO.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5547)
  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 5547)
  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: SITS-ccdb
FEATURES             Location/Qualifiers
     source          1..5547
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    117..137
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     168..187
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        168..186
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             330..353
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     protein_bind    354..477
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        655..757
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             758..1441
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(824..843)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             1761..2066
                     /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     1945..1964
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(2456..2579)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(2587..2603)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    2619..2753
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2643..2662)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2697..2716
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2918..2938)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2918..2936)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2957..2973)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2957..2973)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2970..2992)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2981..2997
                     /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(3005..3011,3012..3029,3030..3035))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3050..3071
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3188..3205)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3359..3947)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3439..3458)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4118..4909,4910..4978))
                     /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     4741..4760
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4979..5083)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5109..5547,1..17))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5246..5267)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5458..5477
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 gccgctacag ggcgcgtccc attcgccatt caggctgcgc aactgttggg aagggcgatc
       61 ggtgcgggcc tcttcgctat tacgccagtc gactagggat aacagggtaa tgaattcaac
      121 tttgtataga aaagttgaca tttccccgaa aagtgccacc tgacgtctaa tacgactcac
      181 tatagggaca tcttaagttt attttatttt attttatttt attttatttt attttatttt
      241 attttatttt attttattta accatgacag taatgtataa agtctgtaaa gacattaaac
      301 acgtaagtga aaccatgggc acctctggtc accaccatca ccaccatcac caccaagttt
      361 gtacaaaaaa gctgaacgag aaacgtaaaa tgatataaat atcaatatat taaattagat
      421 tttgcataaa aaacagacta cataatactg taaaacacaa catatccagt cactatgaat
      481 caactactta gatggtatta gtgacctgta gtcgactaag ttggcagcat cacccgacgc
      541 actttgcgcc gaataaatac ctgtgacgga agatcacttc gcagaataaa taaatcctgg
      601 tgtccctgtt gataccggga agccctgggc caacttttgg cgaaaatgag acgttgatcg
      661 gcacgtaaga ggttccaact ttcaccataa tgaaataaga tcactaccgg gcgtattttt
      721 tgagttatcg agattttcag gagctaagga agctaaaatg gagaaaaaaa tcactggata
      781 taccaccgtt gatatatccc aatggcatcg taaagaacat tttgaggcat ttcagtcagt
      841 tgctcaatgt acctataacc agaccgttca gctggatatt acggcctttt taaagaccgt
      901 aaagaaaaat aagcacaagt tttatccggc ctttattcac attcttgccc gcctgatgaa
      961 tgctcatccg gaattccgta tggcaatgaa agacggtgag ctggtgatat gggatagtgt
     1021 tcacccttgt tacaccgttt tccatgagca aactgaaacg ttttcatcgc tctggagtga
     1081 ataccacgac gatttccggc agtttctaca catatattcg caagatgtgg cgtgttacgg
     1141 tgaaaacctg gcctatttcc ctaaagggtt tattgagaat atgtttttcg tctcagccaa
     1201 tccctgggtg agtttcacca gttttgattt aaacgtggcc aatatggaca acttcttcgc
     1261 ccccgttttc accatgggca aatattatac gcaaggcgac aaggtgctga tgccgctggc
     1321 gattcaggtt catcatgccg tttgtgatgg cttccatgtc ggcagaatgc ttaatgaatt
     1381 acaacagtac tgcgatgagt ggcagggggg gcgtaaacgc cgcgtggatc cggcttacta
     1441 aaagccagat aacagtatgc gtatttgcgc gctgattttt gcggtataag aatatatact
     1501 gatatgtata cccgaagtat gtcaaaaaga ggtatgctat gaagcagcgt attacagtga
     1561 cagttgacag cgacagctat cagttgctca aggcatatat gatgtcaata tctccggtct
     1621 ggtaagcaca accatgcaga atgaagcccg tcgtctgcgt gccgaacgct ggaaagcgga
     1681 aaatcaggaa gggatggctg aggtcgcccg gtttattgaa atgaacggct cttttgctga
     1741 cgagaacagg ggctggtgaa atgcagttta aggtttacac ctataaaaga gagagccgtt
     1801 atcgtctgtt tgtggatgta cagagtgata ttattgacac gcccgggcga cggatggtga
     1861 tccccctggc cagtgcacgt ctgctgtcgg ataaagtctc ccgtgaactt tacccggtgg
     1921 tgcatatcgg ggatgaaagc tggcgcatga tgaccaccga tatggccagt gtgccggtct
     1981 ccgttatcgg ggaagaagtg gctgatctca gccaccgcga aaatgacatc aaaaacgcca
     2041 ttaacctgat gttctgggga atataaatgt caggctccct tatacacagc cagtctgcag
     2101 gtcgatacag tagaaattac agaaacttta tcacgtttag taagtataga ggctgaaaat
     2161 ccagatgaag ccgaacgact tgtaagagaa aagtataaga gttgtgaaat tgttcttgat
     2221 gcagatgatt ttcaggacta tgacactagc gtatatgaat aggtagatgt ttttattttg
     2281 tcacacaaaa aagaggctcg cacctctttt tcttatttct ttttatgatt taatacggca
     2341 ttgaggacaa tagcgagtag gctggatacg acgattccgt ttgagaagaa catttggaag
     2401 gctgtcggtc gagctcgaat tctacaggtc actaatacca tctaagtagt tgattcatag
     2461 tgactgcata tgttgtgttt tacagtatta tgtagtctgt tttttatgca aaatctaatt
     2521 taatatattg atatttatat cattttacgt ttctcgttca actttattat acatagttga
     2581 taattcactg gccgtcgttt tacggtacca tcgatgatga tccagacatg ataagataca
     2641 ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa
     2701 tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca
     2761 acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca
     2821 agtaaaacct ctacaaatgt ggtatggctg attatgatct cgagattacc ctgttatccc
     2881 tagcgtggat cctaggacta gtacccagct tttgttccct ttagtgaggg ttaattgcgc
     2941 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     3001 cacacaacat acgagccggg agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     3061 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     3121 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     3181 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     3241 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     3301 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     3361 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     3421 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     3481 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     3541 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     3601 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     3661 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     3721 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     3781 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     3841 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     3901 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     3961 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     4021 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     4081 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     4141 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     4201 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     4261 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     4321 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     4381 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     4441 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     4501 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     4561 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     4621 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     4681 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     4741 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     4801 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     4861 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     4921 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
     4981 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca
     5041 tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag
     5101 tgccacctaa attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat
     5161 cagctcattt tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata
     5221 gaccgagata gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt
     5281 ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc
     5341 atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa
     5401 agggagcccc cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg
     5461 gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt
     5521 aaccaccaca cccgccgcgc ttaatgc
//
