LOCUS       sequence-341976-        6327 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Multisite gateway destination vector for 3' tagging with EGFP for 
            cell free expression. Parton lab FEX.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6327)
  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 6327)
  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-EGFP
FEATURES             Location/Qualifiers
     source          1..6327
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(43..62)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(44..62)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    complement(93..113)
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     rep_origin      213..668
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(300..319)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     510..531
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        694..798
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(799..867,868..1659)
                     /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(1017..1036)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1830..2418
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2319..2338
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2572..2589
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2706..2727
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2742..2747,2748..2765,2766..2772)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2780..2796
                     /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     2785..2807
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2804..2820
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2804..2820
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     2839..2859
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        2841..2859
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     polyA_signal    3024..3158
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3061..3080)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3115..3134
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3174..3190
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    3198..3218
                     /gene="mutant version of attB"
                     /label=attB3
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     CDS             complement(join(3220..3933,3934..3936))
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             complement(join(3223..3933,3934..3936))
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(3265..3286)
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     3614..3633
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3873..3894
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     protein_bind    3939..4062
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     promoter        4240..4342
                     /label=cat promoter
                     /note="promoter of the E. coli cat gene encoding 
                     chloramphenicol acetyltransferase"
     CDS             4343..5026
                     /codon_start=1
                     /product="chloramphenicol acetyltransferase"
                     /label=CmR
                     /note="confers resistance to chloramphenicol"
                     /translation="MEKKITGYTTVDISQWHRKEHFEAFQSVAQCTYNQTVQLDITAFL
                     KTVKKNKHKFYPAFIHILARLMNAHPEFRMAMKDGELVIWDSVHPCYTVFHEQTETFSS
                     LWSEYHDDFRQFLHIYSQDVACYGENLAYFPKGFIENMFFVSANPWVSFTSFDLNVANM
                     DNFFAPVFTMGKYYTQGDKVLMPLAIQVHHAVCDGFHVGRMLNELQQYCDEWQGGRKRR
                     VDPAY"
     primer_bind     complement(4409..4428)
                     /label=CAT-R
                     /note="Chloramphenicol resistance gene, reverse primer"
     CDS             5346..5651
                     /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     5530..5549
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     protein_bind    complement(6080..6203)
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             complement(6204..6227)
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
ORIGIN
        1 aaataaaata aaataaaata aaataaaata aacttaagat gtccctatag tgagtcgtat
       61 tagacgtcag gtggcacttt tcggggaaat gtcaactttt ctatacaaag ttgaattcat
      121 taccctgtta tccctagtcg actggcgtaa tagcgaagag gcccgcaccg atcgcccttc
      181 ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg cattaagcgc
      241 ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc tagcgcccgc
      301 tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct
      361 aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa
      421 acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc
      481 tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact
      541 caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt cggcctattg
      601 gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgct
      661 tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg tttatttttc
      721 taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat gcttcaataa
      781 tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat tccctttttt
      841 gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct
      901 gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag cggtaagatc
      961 cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa agttctgcta
     1021 tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg ccgcatacac
     1081 tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct tacggatggc
     1141 atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac tgcggccaac
     1201 ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca caacatgggg
     1261 gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat accaaacgac
     1321 gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact attaactggc
     1381 gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc ggataaagtt
     1441 gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga taaatctgga
     1501 gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg taagccctcc
     1561 cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg aaatagacag
     1621 atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca agtttactca
     1681 tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc
     1741 ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca
     1801 gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg cgtaatctgc
     1861 tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta
     1921 ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgttctt
     1981 ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc
     2041 gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg
     2101 ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg
     2161 tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag
     2221 ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc
     2281 agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat
     2341 agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg
     2401 gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc
     2461 tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt
     2521 accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca
     2581 gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc gcgttggccg
     2641 attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag tgagcgcaac
     2701 gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt tatgctcccg
     2761 gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa cagctatgac
     2821 catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct gggtactagt
     2881 cctaggatcc acgctaggga taacagggta atctcgagat cataatcagc cataccacat
     2941 ttgtagaggt tttacttgct ttaaaaaacc tcccacacct ccccctgaac ctgaaacata
     3001 aaatgaatgc aattgttgtt gttaacttgt ttattgcagc ttataatggt tacaaataaa
     3061 gcaatagcat cacaaatttc acaaataaag catttttttc actgcattct agttgtggtt
     3121 tgtccaaact catcaatgta tcttatcatg tctggatcat catcgatggt accgtaaaac
     3181 gacggccagt gaattatcaa ctatgtataa taaagttggt tacttgtaca gctcgtccat
     3241 gccgagagtg atcccggcgg cggtcacgaa ctccagcagg accatgtgat cgcgcttctc
     3301 gttggggtct ttgctcaggg cggactgggt gctcaggtag tggttgtcgg gcagcagcac
     3361 ggggccgtcg ccgatggggg tgttctgctg gtagtggtcg gcgagctgca cgctgccgtc
     3421 ctcgatgttg tggcggatct tgaagttcac cttgatgccg ttcttctgct tgtcggccat
     3481 gatatagacg ttgtggctgt tgtagttgta ctccagcttg tgccccagga tgttgccgtc
     3541 ctccttgaag tcgatgccct tcagctcgat gcggttcacc agggtgtcgc cctcgaactt
     3601 cacctcggcg cgggtcttgt agttgccgtc gtccttgaag aagatggtgc gctcctggac
     3661 gtagccttcg ggcatggcgg acttgaagaa gtcgtgctgc ttcatgtggt cggggtagcg
     3721 gctgaagcac tgcacgccgt aggtcagggt ggtcacgagg gtgggccagg gcacgggcag
     3781 cttgccggtg gtgcagatga acttcagggt cagcttgccg taggtggcat cgccctcgcc
     3841 ctcgccggac acgctgaact tgtggccgtt tacgtcgccg tccagctcga ccaggatggg
     3901 caccaccccg gtgaacagct cctcgccctt gctcacagcc actttgtaca agaaagctga
     3961 acgagaaacg taaaatgata taaatatcaa tatattaaat tagattttgc ataaaaaaca
     4021 gactacataa tactgtaaaa cacaacatat ccagtcacta tgaatcaact acttagatgg
     4081 tattagtgac ctgtagtcga ctaagttggc agcatcaccc gacgcacttt gcgccgaata
     4141 aatacctgtg acggaagatc acttcgcaga ataaataaat cctggtgtcc ctgttgatac
     4201 cgggaagccc tgggccaact tttggcgaaa atgagacgtt gatcggcacg taagaggttc
     4261 caactttcac cataatgaaa taagatcact accgggcgta ttttttgagt tatcgagatt
     4321 ttcaggagct aaggaagcta aaatggagaa aaaaatcact ggatatacca ccgttgatat
     4381 atcccaatgg catcgtaaag aacattttga ggcatttcag tcagttgctc aatgtaccta
     4441 taaccagacc gttcagctgg atattacggc ctttttaaag accgtaaaga aaaataagca
     4501 caagttttat ccggccttta ttcacattct tgcccgcctg atgaatgctc atccggaatt
     4561 ccgtatggca atgaaagacg gtgagctggt gatatgggat agtgttcacc cttgttacac
     4621 cgttttccat gagcaaactg aaacgttttc atcgctctgg agtgaatacc acgacgattt
     4681 ccggcagttt ctacacatat attcgcaaga tgtggcgtgt tacggtgaaa acctggccta
     4741 tttccctaaa gggtttattg agaatatgtt tttcgtctca gccaatccct gggtgagttt
     4801 caccagtttt gatttaaacg tggccaatat ggacaacttc ttcgcccccg ttttcaccat
     4861 gggcaaatat tatacgcaag gcgacaaggt gctgatgccg ctggcgattc aggttcatca
     4921 tgccgtttgt gatggcttcc atgtcggcag aatgcttaat gaattacaac agtactgcga
     4981 tgagtggcag ggggggcgta aacgccgcgt ggatccggct tactaaaagc cagataacag
     5041 tatgcgtatt tgcgcgctga tttttgcggt ataagaatat atactgatat gtatacccga
     5101 agtatgtcaa aaagaggtat gctatgaagc agcgtattac agtgacagtt gacagcgaca
     5161 gctatcagtt gctcaaggca tatatgatgt caatatctcc ggtctggtaa gcacaaccat
     5221 gcagaatgaa gcccgtcgtc tgcgtgccga acgctggaaa gcggaaaatc aggaagggat
     5281 ggctgaggtc gcccggttta ttgaaatgaa cggctctttt gctgacgaga acaggggctg
     5341 gtgaaatgca gtttaaggtt tacacctata aaagagagag ccgttatcgt ctgtttgtgg
     5401 atgtacagag tgatattatt gacacgcccg ggcgacggat ggtgatcccc ctggccagtg
     5461 cacgtctgct gtcagataaa gtctcccgtg aactttaccc ggtggtgcat atcggggatg
     5521 aaagctggcg catgatgacc accgatatgg ccagtgtgcc ggtctccgtt atcggggaag
     5581 aagtggctga tctcagccac cgcgaaaatg acatcaaaaa cgccattaac ctgatgttct
     5641 ggggaatata aatgtcaggc tcccttatac acagccagtc tgcaggtcga tacagtagaa
     5701 attacagaaa ctttatcacg tttagtaagt atagaggctg aaaatccaga tgaagccgaa
     5761 cgacttgtaa gagaaaagta taagagttgt gaaattgttc ttgatgcaga tgattttcag
     5821 gactatgaca ctagcgtata tgaataggta gatgttttta ttttgtcaca caaaaaagag
     5881 gctcgcacct ctttttctta tttcttttta tgatttaata cggcattgag gacaatagcg
     5941 agtaggctgg atacgacgat tccgtttgag aagaacattt ggaaggctgt cggtcgacta
     6001 agttggcagc atcacccgaa gaacatttgg aaggctgtcg gtcgactaca ggtcactaat
     6061 accatctaag tagttgattc atagtgactg gatatgttgt gttttacagt attatgtagt
     6121 ctgtttttta tgcaaaatct aatttaatat attgatattt atatcatttt acgtttctcg
     6181 ttcagctttt ttgtacaaac ttggtggtga tggtggtgat ggtggtgacc agaggtgccc
     6241 atggtttcac ttacgtgttt aatgtcttta cagactttat acattactgt catggttaaa
     6301 taaaataaaa taaaataaaa taaaata
//
