LOCUS       sequence-363983-        6318 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite gateway destination vector for 3' tagging with mCherry for
            cell free expression. Parton lab clone FFF.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6318)
  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 6318)
  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-mCherry
FEATURES             Location/Qualifiers
     source          1..6318
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    116..136
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     167..186
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        167..185
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             329..352
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     protein_bind    353..476
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(905..1210)
                     /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(1007..1026)
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             complement(1530..2213)
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     2128..2147
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     promoter        complement(2214..2316)
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     protein_bind    complement(2494..2617)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             2620..3327
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(2762..2780)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     3030..3049
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     3224..3247
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(3329..3349)
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     complement(3357..3373)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    3389..3523
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3413..3432)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3467..3486
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3688..3708)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3688..3706)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3727..3743)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3727..3743)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3740..3762)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3751..3767
                     /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(3775..3781,3782..3799,3800..3805))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3820..3841
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3958..3975)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4129..4717)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4209..4228)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4888..5679,5680..5748))
                     /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     5511..5530
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5749..5853)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(join(5879..6318,1..16))
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(6016..6037)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6228..6247
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 ccgctacagg gcgcgtccca ttcgccattc aggctgcgca actgttggga agggcgatcg
       61 gtgcgggcct cttcgctatt acgccagtcg actagggata acagggtaat gaattcaact
      121 ttgtatagaa aagttgacat ttccccgaaa agtgccacct gacgtctaat acgactcact
      181 atagggacat cttaagttta ttttatttta ttttatttta ttttatttta ttttatttta
      241 ttttatttta ttttatttaa ccatgacagt aatgtataaa gtctgtaaag acattaaaca
      301 cgtaagtgaa accatgggca cctctggtca ccaccatcac caccatcacc accaagtttg
      361 tacaaaaaag ctgaacgaga aacgtaaaat gatataaata tcaatatatt aaattagatt
      421 ttgcataaaa aacagactac ataatactgt aaaacacaac atatccagtc actatgaatc
      481 aactacttag atggtattag tgacctgtag tcgaccgaca gccttccaaa tgttcttcgg
      541 gtgatgctgc caacttagtc gaccgacagc cttccaaatg ttcttctcaa acggaatcgt
      601 cgtatccagc ctactcgcta ttgtcctcaa tgccgtatta aatcataaaa agaaataaga
      661 aaaagaggtg cgagcctctt ttttgtgtga caaaataaaa acatctacct attcatatac
      721 gctagtgtca tagtcctgaa aatcatctgc atcaagaaca atttcacaac tcttatactt
      781 ttctcttaca agtcgttcgg cttcatctgg attttcagcc tctatactta ctaaacgtga
      841 taaagtttct gtaatttcta ctgtatcgac ctgcagactg gctgtgtata agggagcctg
      901 acatttatat tccccagaac atcaggttaa tggcgttttt gatgtcattt tcgcggtggc
      961 tgagatcagc cacttcttcc ccgataacgg agaccggcac actggccata tcggtggtca
     1021 tcatgcgcca gctttcatcc ccgatatgca ccaccgggta aagttcacgg gagactttat
     1081 ctgacagcag acgtgcactg gccaggggga tcaccatccg tcgcccgggc gtgtcaataa
     1141 tatcactctg tacatccaca aacagacgat aacggctctc tcttttatag gtgtaaacct
     1201 taaactgcat ttcaccagcc cctgttctcg tcagcaaaag agccgttcat ttcaataaac
     1261 cgggcgacct cagccatccc ttcctgattt tccgctttcc agcgttcggc acgcagacga
     1321 cgggcttcat tctgcatggt tgtgcttacc agaccggaga tattgacatc atatatgcct
     1381 tgagcaactg atagctgtcg ctgtcaactg tcactgtaat acgctgcttc atagcatacc
     1441 tctttttgac atacttcggg tatacatatc agtatatatt cttataccgc aaaaatcagc
     1501 gcgcaaatac gcatactgtt atctggcttt tagtaagccg gatccacgcg gcgtttacgc
     1561 cccccctgcc actcatcgca gtactgttgt aattcattaa gcattctgcc gacatggaag
     1621 ccatcacaaa cggcatgatg aacctgaatc gccagcggca tcagcacctt gtcgccttgc
     1681 gtataatatt tgcccatggt gaaaacgggg gcgaagaagt tgtccatatt ggccacgttt
     1741 aaatcaaaac tggtgaaact cacccaggga ttggctgaga cgaaaaacat attctcaata
     1801 aaccctttag ggaaataggc caggttttca ccgtaacacg ccacatcttg cgaatatatg
     1861 tgtagaaact gccggaaatc gtcgtggtat tcactccaga gcgatgaaaa cgtttcagtt
     1921 tgctcatgga aaacggtgta acaagggtga acactatccc atatcaccag ctcaccgtct
     1981 ttcattgcca tacggaattc cggatgagca ttcatcaggc gggcaagaat gtgaataaag
     2041 gccggataaa acttgtgctt atttttcttt acggtcttta aaaaggccgt aatatccagc
     2101 tgaacggtct ggttataggt acattgagca actgactgaa atgcctcaaa atgttcttta
     2161 cgatgccatt gggatatatc aacggtggta tatccagtga tttttttctc cattttagct
     2221 tccttagctc ctgaaaatct cgataactca aaaaatacgc ccggtagtga tcttatttca
     2281 ttatggtgaa agttggaacc tcttacgtgc cgatcaacgt ctcattttcg ccaaaagttg
     2341 gcccagggct tcccggtatc aacagggaca ccaggattta tttattctgc gaagtgatct
     2401 tccgtcacag gtatttattc ggcgcaaagt gcgtcgggtg atgctgccaa cttagtcgac
     2461 tacaggtcac taataccatc taagtagttg attcatagtg actggatatg ttgtgtttta
     2521 cagtattatg tagtctgttt tttatgcaaa atctaattta atatattgat atttatatca
     2581 ttttacgttt ctcgttcagc tttcttgtac aaagtggctg tgagcaaggg cgaggaggac
     2641 aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
     2701 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
     2761 gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtcccct
     2821 cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg
     2881 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
     2941 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
     3001 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg
     3061 gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag
     3121 aggctgaagc tgaaggacgg cggccactac gacgccgagg tcaagaccac ctacaaggcc
     3181 aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagctgga catcacctcc
     3241 cacaacgagg actacaccat cgtggaacag tacgagcgcg ccgagggccg ccactccacc
     3301 ggcggcatgg acgagctgta caagtaacca actttattat acatagttga taattcactg
     3361 gccgtcgttt tacggtacca tcgatgatga tccagacatg ataagataca ttgatgagtt
     3421 tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt atttgtgaaa tttgtgatgc
     3481 tattgcttta tttgtaacca ttataagctg caataaacaa gttaacaaca acaattgcat
     3541 tcattttatg tttcaggttc agggggaggt gtgggaggtt ttttaaagca agtaaaacct
     3601 ctacaaatgt ggtatggctg attatgatct cgagattacc ctgttatccc tagcgtggat
     3661 cctaggacta gtacccagct tttgttccct ttagtgaggg ttaattgcgc gcttggcgta
     3721 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     3781 acgagccggg agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     3841 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta
     3901 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     3961 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4021 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     4081 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     4141 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     4201 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     4261 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     4321 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     4381 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     4441 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     4501 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     4561 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     4621 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     4681 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     4741 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     4801 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     4861 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     4921 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     4981 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     5041 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     5101 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     5161 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     5221 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     5281 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     5341 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     5401 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     5461 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     5521 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     5581 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     5641 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     5701 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     5761 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     5821 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctaa
     5881 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     5941 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     6001 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     6061 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     6121 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     6181 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     6241 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     6301 cccgccgcgc ttaatgcg
//
