LOCUS       sequence-341968-        6236 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite gateway destination vector for 5' tagging with mCherry for
            cell free expression. Parton lab clone FKZ.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6236)
  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 6236)
  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-mCherry-ccdb
FEATURES             Location/Qualifiers
     source          1..6236
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    100..120
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     primer_bind     151..170
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        151..169
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             308..331
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     protein_bind    331..355
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      356..365
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             362..1069
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEG
                     TQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNF
                     EDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALK
                     GEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERA
                     EGRHSTGGMDELYK"
     primer_bind     complement(507..525)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     775..794
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     969..992
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    complement(1071..1170)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1327..1429
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1430..2113
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(1496..1515)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2433..2738
                     /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     2617..2636
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3128..3251)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3259..3275)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    3291..3425
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3315..3334)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3369..3388
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3590..3610)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3590..3608)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3629..3645)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3629..3645)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3642..3664)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3653..3669
                     /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(3677..3683,3684..3701,3702..3707))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3722..3743
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3860..3877)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4031..4619)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4111..4130)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4790..5581,5582..5650))
                     /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     5413..5432
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5651..5755)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5781..6236)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5918..5939)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6130..6149
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
ORIGIN
        1 cccattcgcc attcaggctg cgcaactgtt gggaagggcg atcggtgcgg gcctcttcgc
       61 tattacgcca gtcgactagg gataacaggg taatgaattc aactttgtat agaaaagttg
      121 acatttcccc gaaaagtgcc acctgacgtc taatacgact cactataggg acatcttaag
      181 tttattttat tttattttat tttattttat tttattttat tttattttat tttatttaac
      241 catgacagta atgtataaag tctgtaaaga cattaaacac gtaagtgaaa ccatgggcac
      301 ctctggtcac caccatcacc accatcacca ccaagtttgt acaaaaaagc aggctgccac
      361 catggtgagc aagggcgagg aggacaacat ggccatcatc aaggagttca tgcgcttcaa
      421 ggtgcacatg gagggctccg tgaacggcca cgagttcgag atcgagggcg agggcgaggg
      481 ccgcccctac gagggcaccc agaccgccaa gctgaaggtg accaagggcg gccccctgcc
      541 cttcgcctgg gacatcctgt cccctcagtt catgtacggc tccaaggcct acgtgaagca
      601 ccccgccgac atccccgact acttgaagct gtccttcccc gagggcttca agtgggagcg
      661 cgtgatgaac ttcgaggacg gcggcgtggt gaccgtgacc caggactcct ccctgcagga
      721 cggcgagttc atctacaagg tgaagctgcg cggcaccaac ttcccctccg acggccccgt
      781 aatgcagaag aagaccatgg gctgggaggc ctcctccgag cggatgtacc ccgaggacgg
      841 cgccctgaag ggcgagatca agcagaggct gaagctgaag gacggcggcc actacgacgc
      901 cgaggtcaag accacctaca aggccaagaa gcccgtgcag ctgcccggcg cctacaacgt
      961 caacatcaag ctggacatca cctcccacaa cgaggactac accatcgtgg aacagtacga
     1021 gcgcgccgag ggccgccact ccaccggcgg catggacgag ctgtacaagc acccagcttt
     1081 cttgtacaaa gttggcatta taaaaaagca ttgcttatca atttgttgca acgaacaggt
     1141 cactatcagt caaaataaaa tcattatttg gagctctaga gcgtcgacta agttggcagc
     1201 atcacccgac gcactttgcg ccgaataaat acctgtgacg gaagatcact tcgcagaata
     1261 aataaatcct ggtgtccctg ttgataccgg gaagccctgg gccaactttt ggcgaaaatg
     1321 agacgttgat cggcacgtaa gaggttccaa ctttcaccat aatgaaataa gatcactacc
     1381 gggcgtattt tttgagttat cgagattttc aggagctaag gaagctaaaa tggagaaaaa
     1441 aatcactgga tataccaccg ttgatatatc ccaatggcat cgtaaagaac attttgaggc
     1501 atttcagtca gttgctcaat gtacctataa ccagaccgtt cagctggata ttacggcctt
     1561 tttaaagacc gtaaagaaaa ataagcacaa gttttatccg gcctttattc acattcttgc
     1621 ccgcctgatg aatgctcatc cggaattccg tatggcaatg aaagacggtg agctggtgat
     1681 atgggatagt gttcaccctt gttacaccgt tttccatgag caaactgaaa cgttttcatc
     1741 gctctggagt gaataccacg acgatttccg gcagtttcta cacatatatt cgcaagatgt
     1801 ggcgtgttac ggtgaaaacc tggcctattt ccctaaaggg tttattgaga atatgttttt
     1861 cgtctcagcc aatccctggg tgagtttcac cagttttgat ttaaacgtgg ccaatatgga
     1921 caacttcttc gcccccgttt tcaccatggg caaatattat acgcaaggcg acaaggtgct
     1981 gatgccgctg gcgattcagg ttcatcatgc cgtttgtgat ggcttccatg tcggcagaat
     2041 gcttaatgaa ttacaacagt actgcgatga gtggcagggg gggcgtaaac gccgcgtgga
     2101 tccggcttac taaaagccag ataacagtat gcgtatttgc gcgctgattt ttgcggtata
     2161 agaatatata ctgatatgta tacccgaagt atgtcaaaaa gaggtatgct atgaagcagc
     2221 gtattacagt gacagttgac agcgacagct atcagttgct caaggcatat atgatgtcaa
     2281 tatctccggt ctggtaagca caaccatgca gaatgaagcc cgtcgtctgc gtgccgaacg
     2341 ctggaaagcg gaaaatcagg aagggatggc tgaggtcgcc cggtttattg aaatgaacgg
     2401 ctcttttgct gacgagaaca ggggctggtg aaatgcagtt taaggtttac acctataaaa
     2461 gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac acgcccgggc
     2521 gacggatggt gatccccctg gccagtgcac gtctgctgtc ggataaagtc tcccgtgaac
     2581 tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc gatatggcca
     2641 gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc gaaaatgaca
     2701 tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggctcc cttatacaca
     2761 gccagtctgc aggtcgatac agtagaaatt acagaaactt tatcacgttt agtaagtata
     2821 gaggctgaaa atccagatga agccgaacga cttgtaagag aaaagtataa gagttgtgaa
     2881 attgttcttg atgcagatga ttttcaggac tatgacacta gcgtatatga ataggtagat
     2941 gtttttattt tgtcacacaa aaaagaggct cgcacctctt tttcttattt ctttttatga
     3001 tttaatacgg cattgaggac aatagcgagt aggctggata cgacgattcc gtttgagaag
     3061 aacatttgga aggctgtcgg tcgagctcga attctacagg tcactaatac catctaagta
     3121 gttgattcat agtgactgca tatgttgtgt tttacagtat tatgtagtct gttttttatg
     3181 caaaatctaa tttaatatat tgatatttat atcattttac gtttctcgtt caactttatt
     3241 atacatagtt gataattcac tggccgtcgt tttacggtac catcgatgat gatccagaca
     3301 tgataagata cattgatgag tttggacaaa ccacaactag aatgcagtga aaaaaatgct
     3361 ttatttgtga aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac
     3421 aagttaacaa caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg
     3481 ttttttaaag caagtaaaac ctctacaaat gtggtatggc tgattatgat ctcgagatta
     3541 ccctgttatc cctagcgtgg atcctaggac tagtacccag cttttgttcc ctttagtgag
     3601 ggttaattgc gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc
     3661 cgctcacaat tccacacaac atacgagccg ggagcataaa gtgtaaagcc tggggtgcct
     3721 aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa
     3781 acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta
     3841 ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc
     3901 gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg
     3961 caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt
     4021 tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa
     4081 gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct
     4141 ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc
     4201 cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg
     4261 tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct
     4321 tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag
     4381 cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga
     4441 agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga
     4501 agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg
     4561 gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag
     4621 aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag
     4681 ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat
     4741 gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct
     4801 taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac
     4861 tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa
     4921 tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg
     4981 gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt
     5041 gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca
     5101 ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt
     5161 cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct
     5221 tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg
     5281 cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg
     5341 agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg
     5401 cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa
     5461 aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt
     5521 aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt
     5581 gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt
     5641 gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca
     5701 tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat
     5761 ttccccgaaa agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa
     5821 tttttgttaa atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa
     5881 atcaaaagaa tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact
     5941 attaaagaac gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc
     6001 actacgtgaa ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa
     6061 tcggaaccct aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc
     6121 gagaaaggaa gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt
     6181 cacgctgcgc gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgt
//
