LOCUS       sequence-325145-        3359 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  126573: Multisite Gateway middle entry vector for fusing mScarlet to
            the 3' end of an ORF..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3359)
  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 3359)
  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: p3E-EGFP
FEATURES             Location/Qualifiers
     source          1..3359
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     27..44
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(203..230)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(322..408)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     457..479
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     471..488
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     472..488
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    complement(559..682)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             join(685..687,688..1401)
                     /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             join(685..687,688..1398)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(727..748)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(988..1007)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1335..1356
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(1403..1498)
                     /gene="mutant version of attL"
                     /label=attL3
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     primer_bind     complement(1535..1554)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1536..1554)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1559..1575)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1559..1575)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(1677..1696)
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     CDS             1688..2497
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYGKP
                     DAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGKTA
                     FQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDASD
                     FDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGIAD
                     RYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     complement(1755..1774)
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     rep_origin      2644..3232
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3133..3152
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg gaagagcgcc
       61 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
      121 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatacgcgt accgctagcc
      181 aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg atggccttct gcttagtttg
      241 atgcctggca gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt gcttcacaac
      301 gttcaaatcc gctcccggcg gatttgtcct actcaggaga gcgttcaccg acaaacaaca
      361 gataaaacga aaggcccagt cttccgactg agcctttcgt tttatttgat gcctggcagt
      421 tccctactct cgcgttaacg ctagcatgga tgttttccca gtcacgacgt tgtaaaacga
      481 cggccagtct taagctcggg ccctgcagct ctagagctcg aattctacag gtcactaata
      541 ccatctaagt agttggttca tagtgactgc atatgttgtg ttttacagta ttatgtagtc
      601 tgttttttat gcaaaatcta atttaatata ttgatattta tatcatttta cgtttctcgt
      661 tcaactttct tgtacaaagt ggctgtgagc aagggcgagg agctgttcac cggggtggtg
      721 cccatcctgg tcgagctgga cggcgacgta aacggccaca agttcagcgt gtccggcgag
      781 ggcgagggcg atgccaccta cggcaagctg accctgaagt tcatctgcac caccggcaag
      841 ctgcccgtgc cctggcccac cctcgtgacc accctgacct acggcgtgca gtgcttcagc
      901 cgctaccccg accacatgaa gcagcacgac ttcttcaagt ccgccatgcc cgaaggctac
      961 gtccaggagc gcaccatctt cttcaaggac gacggcaact acaagacccg cgccgaggtg
     1021 aagttcgagg gcgacaccct ggtgaaccgc atcgagctga agggcatcga cttcaaggag
     1081 gacggcaaca tcctggggca caagctggag tacaactaca acagccacaa cgtctatatc
     1141 atggccgaca agcagaagaa cggcatcaag gtgaacttca agatccgcca caacatcgag
     1201 gacggcagcg tgcagctcgc cgaccactac cagcagaaca cccccatcgg cgacggcccc
     1261 gtgctgctgc ccgacaacca ctacctgagc acccagtccg ccctgagcaa agaccccaac
     1321 gagaagcgcg atcacatggt cctgctggag ttcgtgaccg ccgccgggat cactctcggc
     1381 atggacgagc tgtacaagta accaacttta ttatacaaag ttggcattat aaaaaagcat
     1441 tgcttatcaa tttgttgcaa cgaacaggtc actatcagtc aaaataaaat cattatttgg
     1501 agctccatgg tagcgttaac gcggccgcga tatcccctat agtgagtcgt attacatggt
     1561 catagctgtt tcctggcagc tctggcccgt gtctcaaaat ctctgatgtt acattgcaca
     1621 agataaaaat atatcatcat gaacaataaa actgtctgct tacataaaca gtaatacaag
     1681 gggtgttatg agccatattc aacgggaaac gtcgaggccg cgattaaatt ccaacatgga
     1741 tgctgattta tatgggtata aatgggctcg cgataatgtc gggcaatcag gtgcgacaat
     1801 ctatcgcttg tatgggaagc ccgatgcgcc agagttgttt ctgaaacatg gcaaaggtag
     1861 cgttgccaat gatgttacag atgagatggt cagactaaac tggctgacgg aatttatgcc
     1921 tcttccgacc atcaagcatt ttatccgtac tcctgatgat gcatggttac tcaccactgc
     1981 gatccccgga aaaacagcat tccaggtatt agaagaatat cctgattcag gtgaaaatat
     2041 tgttgatgcg ctggcagtgt tcctgcgccg gttgcattcg attcctgttt gtaattgtcc
     2101 ttttaacagc gatcgcgtat ttcgtctcgc tcaggcgcaa tcacgaatga ataacggttt
     2161 ggttgatgcg agtgattttg atgacgagcg taatggctgg cctgttgaac aagtctggaa
     2221 agaaatgcat aaacttttgc cattctcacc ggattcagtc gtcactcatg gtgatttctc
     2281 acttgataac cttatttttg acgaggggaa attaataggt tgtattgatg ttggacgagt
     2341 cggaatcgca gaccgatacc aggatcttgc catcctatgg aactgcctcg gtgagttttc
     2401 tccttcatta cagaaacggc tttttcaaaa atatggtatt gataatcctg atatgaataa
     2461 attgcagttt catttgatgc tcgatgagtt tttctaatca gaattggtta attggttgta
     2521 acactggcag agcattacgc tgacttgacg ggacggcgca agctcatgac caaaatccct
     2581 taacgtgagt tacgcgtcgt tccactgagc gtcagacccc gtagaaaaga tcaaaggatc
     2641 ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa aaccaccgct
     2701 accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga aggtaactgg
     2761 cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt taggccacca
     2821 cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt taccagtggc
     2881 tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat agttaccgga
     2941 taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct tggagcgaac
     3001 gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca cgcttcccga
     3061 agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag agcgcacgag
     3121 ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc gccacctctg
     3181 acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga aaaacgccag
     3241 caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca tgttctttcc
     3301 tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag ctgataccg
//
