LOCUS       sequence-341964-        6250 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite gateway destination vector for 5' tagging with EGFP for 
            cell free expression. Parton lab clone FEZ.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6250)
  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 6250)
  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-EGFP-ccdb
FEATURES             Location/Qualifiers
     source          1..6250
                     /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             313..336
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     protein_bind    336..360
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      361..370
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(367..369,370..372,373..1083)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(367..369,370..372,373..1083)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(412..433)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(673..692)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1020..1041
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1085..1184)
                     /gene="mutant version of attL"
                     /label=attL2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        1341..1443
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             1444..2127
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(1510..1529)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             2447..2752
                     /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     2631..2650
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(3142..3265)
                     /gene="mutant version of attR"
                     /label=attR3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(3273..3289)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    3305..3439
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3329..3348)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3383..3402
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(3604..3624)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(3604..3622)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(3643..3659)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3643..3659)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3656..3678)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3667..3683
                     /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(3691..3697,3698..3715,3716..3721))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3736..3757
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3874..3891)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4045..4633)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4125..4144)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4804..5595,5596..5664))
                     /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     5427..5446
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5665..5769)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(5795..6250)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5932..5953)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     6144..6163
                     /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 tttattttat
      241 ttaaccatga cagtaatgta taaagtctgt aaagacatta aacacgtaag tgaaaccatg
      301 ggcacctctg gtcaccacca tcaccaccat caccaccaag tttgtacaaa aaagcaggct
      361 gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat cctggtcgag
      421 ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga gggcgatgcc
      481 acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc cgtgccctgg
      541 cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta ccccgaccac
      601 atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca ggagcgcacc
      661 atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt cgagggcgac
      721 accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg caacatcctg
      781 gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc cgacaagcag
      841 aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg cagcgtgcag
      901 ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct gctgcccgac
      961 aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa gcgcgatcac
     1021 atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga cgagctgtac
     1081 aagcacccag ctttcttgta caaagttggc attataaaaa agcattgctt atcaatttgt
     1141 tgcaacgaac aggtcactat cagtcaaaat aaaatcatta tttggagctc tagagcgtcg
     1201 actaagttgg cagcatcacc cgacgcactt tgcgccgaat aaatacctgt gacggaagat
     1261 cacttcgcag aataaataaa tcctggtgtc cctgttgata ccgggaagcc ctgggccaac
     1321 ttttggcgaa aatgagacgt tgatcggcac gtaagaggtt ccaactttca ccataatgaa
     1381 ataagatcac taccgggcgt attttttgag ttatcgagat tttcaggagc taaggaagct
     1441 aaaatggaga aaaaaatcac tggatatacc accgttgata tatcccaatg gcatcgtaaa
     1501 gaacattttg aggcatttca gtcagttgct caatgtacct ataaccagac cgttcagctg
     1561 gatattacgg cctttttaaa gaccgtaaag aaaaataagc acaagtttta tccggccttt
     1621 attcacattc ttgcccgcct gatgaatgct catccggaat tccgtatggc aatgaaagac
     1681 ggtgagctgg tgatatggga tagtgttcac ccttgttaca ccgttttcca tgagcaaact
     1741 gaaacgtttt catcgctctg gagtgaatac cacgacgatt tccggcagtt tctacacata
     1801 tattcgcaag atgtggcgtg ttacggtgaa aacctggcct atttccctaa agggtttatt
     1861 gagaatatgt ttttcgtctc agccaatccc tgggtgagtt tcaccagttt tgatttaaac
     1921 gtggccaata tggacaactt cttcgccccc gttttcacca tgggcaaata ttatacgcaa
     1981 ggcgacaagg tgctgatgcc gctggcgatt caggttcatc atgccgtttg tgatggcttc
     2041 catgtcggca gaatgcttaa tgaattacaa cagtactgcg atgagtggca gggggggcgt
     2101 aaacgccgcg tggatccggc ttactaaaag ccagataaca gtatgcgtat ttgcgcgctg
     2161 atttttgcgg tataagaata tatactgata tgtatacccg aagtatgtca aaaagaggta
     2221 tgctatgaag cagcgtatta cagtgacagt tgacagcgac agctatcagt tgctcaaggc
     2281 atatatgatg tcaatatctc cggtctggta agcacaacca tgcagaatga agcccgtcgt
     2341 ctgcgtgccg aacgctggaa agcggaaaat caggaaggga tggctgaggt cgcccggttt
     2401 attgaaatga acggctcttt tgctgacgag aacaggggct ggtgaaatgc agtttaaggt
     2461 ttacacctat aaaagagaga gccgttatcg tctgtttgtg gatgtacaga gtgatattat
     2521 tgacacgccc gggcgacgga tggtgatccc cctggccagt gcacgtctgc tgtcggataa
     2581 agtctcccgt gaactttacc cggtggtgca tatcggggat gaaagctggc gcatgatgac
     2641 caccgatatg gccagtgtgc cggtctccgt tatcggggaa gaagtggctg atctcagcca
     2701 ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc tggggaatat aaatgtcagg
     2761 ctcccttata cacagccagt ctgcaggtcg atacagtaga aattacagaa actttatcac
     2821 gtttagtaag tatagaggct gaaaatccag atgaagccga acgacttgta agagaaaagt
     2881 ataagagttg tgaaattgtt cttgatgcag atgattttca ggactatgac actagcgtat
     2941 atgaataggt agatgttttt attttgtcac acaaaaaaga ggctcgcacc tctttttctt
     3001 atttcttttt atgatttaat acggcattga ggacaatagc gagtaggctg gatacgacga
     3061 ttccgtttga gaagaacatt tggaaggctg tcggtcgagc tcgaattcta caggtcacta
     3121 ataccatcta agtagttgat tcatagtgac tgcatatgtt gtgttttaca gtattatgta
     3181 gtctgttttt tatgcaaaat ctaatttaat atattgatat ttatatcatt ttacgtttct
     3241 cgttcaactt tattatacat agttgataat tcactggccg tcgttttacg gtaccatcga
     3301 tgatgatcca gacatgataa gatacattga tgagtttgga caaaccacaa ctagaatgca
     3361 gtgaaaaaaa tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat
     3421 aagctgcaat aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
     3481 ggaggtgtgg gaggtttttt aaagcaagta aaacctctac aaatgtggta tggctgatta
     3541 tgatctcgag attaccctgt tatccctagc gtggatccta ggactagtac ccagcttttg
     3601 ttccctttag tgagggttaa ttgcgcgctt ggcgtaatca tggtcatagc tgtttcctgt
     3661 gtgaaattgt tatccgctca caattccaca caacatacga gccgggagca taaagtgtaa
     3721 agcctggggt gcctaatgag tgagctaact cacattaatt gcgttgcgct cactgcccgc
     3781 tttccagtcg ggaaacctgt cgtgccagct gcattaatga atcggccaac gcgcggggag
     3841 aggcggtttg cgtattgggc gctcttccgc ttcctcgctc actgactcgc tgcgctcggt
     3901 cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg gtaatacggt tatccacaga
     3961 atcaggggat aacgcaggaa agaacatgtg agcaaaaggc cagcaaaagg ccaggaaccg
     4021 taaaaaggcc gcgttgctgg cgtttttcca taggctccgc ccccctgacg agcatcacaa
     4081 aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga ctataaagat accaggcgtt
     4141 tccccctgga agctccctcg tgcgctctcc tgttccgacc ctgccgctta ccggatacct
     4201 gtccgccttt ctcccttcgg gaagcgtggc gctttctcat agctcacgct gtaggtatct
     4261 cagttcggtg taggtcgttc gctccaagct gggctgtgtg cacgaacccc ccgttcagcc
     4321 cgaccgctgc gccttatccg gtaactatcg tcttgagtcc aacccggtaa gacacgactt
     4381 atcgccactg gcagcagcca ctggtaacag gattagcaga gcgaggtatg taggcggtgc
     4441 tacagagttc ttgaagtggt ggcctaacta cggctacact agaagaacag tatttggtat
     4501 ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt ggtagctctt gatccggcaa
     4561 acaaaccacc gctggtagcg gtggtttttt tgtttgcaag cagcagatta cgcgcagaaa
     4621 aaaaggatct caagaagatc ctttgatctt ttctacgggg tctgacgctc agtggaacga
     4681 aaactcacgt taagggattt tggtcatgag attatcaaaa aggatcttca cctagatcct
     4741 tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata tatgagtaaa cttggtctga
     4801 cagttaccaa tgcttaatca gtgaggcacc tatctcagcg atctgtctat ttcgttcatc
     4861 catagttgcc tgactccccg tcgtgtagat aactacgata cgggagggct taccatctgg
     4921 ccccagtgct gcaatgatac cgcgagaccc acgctcaccg gctccagatt tatcagcaat
     4981 aaaccagcca gccggaaggg ccgagcgcag aagtggtcct gcaactttat ccgcctccat
     5041 ccagtctatt aattgttgcc gggaagctag agtaagtagt tcgccagtta atagtttgcg
     5101 caacgttgtt gccattgcta caggcatcgt ggtgtcacgc tcgtcgtttg gtatggcttc
     5161 attcagctcc ggttcccaac gatcaaggcg agttacatga tcccccatgt tgtgcaaaaa
     5221 agcggttagc tccttcggtc ctccgatcgt tgtcagaagt aagttggccg cagtgttatc
     5281 actcatggtt atggcagcac tgcataattc tcttactgtc atgccatccg taagatgctt
     5341 ttctgtgact ggtgagtact caaccaagtc attctgagaa tagtgtatgc ggcgaccgag
     5401 ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca catagcagaa ctttaaaagt
     5461 gctcatcatt ggaaaacgtt cttcggggcg aaaactctca aggatcttac cgctgttgag
     5521 atccagttcg atgtaaccca ctcgtgcacc caactgatct tcagcatctt ttactttcac
     5581 cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc gcaaaaaagg gaataagggc
     5641 gacacggaaa tgttgaatac tcatactctt cctttttcaa tattattgaa gcatttatca
     5701 gggttattgt ctcatgagcg gatacatatt tgaatgtatt tagaaaaata aacaaatagg
     5761 ggttccgcgc acatttcccc gaaaagtgcc acctaaattg taagcgttaa tattttgtta
     5821 aaattcgcgt taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc
     5881 aaaatccctt ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg
     5941 aacaagagtc cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat
     6001 cagggcgatg gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc
     6061 cgtaaagcac taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag
     6121 ccggcgaacg tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg
     6181 gcaagtgtag cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta
     6241 cagggcgcgt
//
