LOCUS       sequence-495428-        6840 bp    DNA     circular SYN 17-DEC-2025
DEFINITION  An EF1a driven expression vector containing an EGFP fused to a 
            microRNA multiple cloning site. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6840)
  AUTHORS   Gillespie WB 3rd, Zhang Y, Ruiz OE, Cerda J 3rd, Ortiz-Guzman J, 
            Sherman M, Turner WD, Largoza G, Mosser LE, Fujimoto E, Chien CB, 
            Kwan KM, Arenkiel BR, Devine WP, Wythe JD
  TITLE     MultiSite Assembly of Gateway Induced Clones (MAGIC): a flexible 
            cloning toolbox for use in vertebrate model systems.
  JOURNAL   Development. 2025 Nov 15;152(22):dev204308. doi: 10.1242/dev.204308.
            Epub 2025 Nov 13.
  PUBMED    41230856
REFERENCE   2  (bases 1 to 6840)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 17, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Development."; date: "2025-11-15"; volume: "152(22):dev204308. 
            doi"; pages: " 10.1242/dev.204308. Epub 2025 Nov 13"
COMMENT     Sequence Label: pEF1a-EGFP-miR-MCS (JDW 1478)
FEATURES             Location/Qualifiers
     source          1..6840
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(166..185)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        473..1651
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          704..1642
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1599..1619
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1668..1687
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1668..1686
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1718..1742
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      1774..1783
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1780..1782,1783..1785,1786..2244)
                     /codon_start=1
                     /product="improved N-terminal fragment of mVenus for use in
                     bimolecular fluorescence complementation (BiFC) (Kodama and
                     Hu, 2010)"
                     /label=VN155(I152L)
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMA"
     primer_bind     complement(1825..1846)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2086..2105)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2692..2713
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     protein_bind    complement(2788..2812)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2838..2879
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2889..2906
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(2929..2946)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2935..3159
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      3205..3633
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3292..3311)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3502..3523
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3642..3662)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        3647..3976
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      3827..3962
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     3889..3908
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     promoter        4024..4071
                     /label=EM7 promoter
                     /note="synthetic bacterial promoter "
     CDS             4090..4488
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    4646..4767
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4683..4702)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4737..4756
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4816..4832)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4816..4832)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4829..4851)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4840..4856
                     /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(4864..4870,4871..4888,4889..4894))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4909..4930
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5047..5064)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5207..5795)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5287..5306)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5966..6757,6758..6826))
                     /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     6589..6608
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(join(6827..6840,1..91))
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
       61 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
      121 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
      181 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
      241 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
      301 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
      361 ttgattattg actaggcttt tgcaaaaagc tttgcaaaga tggataaagt tttaaacaga
      421 gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa ttggctccgg
      481 tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt
      541 cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt
      601 gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc
      661 cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg
      721 ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac
      781 ctggctgcag tacgtgattc ttgatcccga gcttcgggtt ggaagtgggt gggagagttc
      841 gaggccttgc gcttaaggag ccccttcgcc tcgtgcttga gttgaggcct ggcctgggcg
      901 ctggggccgc cgcgtgcgaa tctggtggca ccttcgcgcc tgtctcgctg ctttcgataa
      961 gtctctagcc atttaaaatt tttgatgacc tgctgcgacg ctttttttct ggcaagatag
     1021 tcttgtaaat gcgggccaag atctgcacac tggtatttcg gtttttgggg ccgcgggcgg
     1081 cgacggggcc cgtgcgtccc agcgcacatg ttcggcgagg cggggcctgc gagcgcggcc
     1141 accgagaatc ggacgggggt agtctcaagc tggccggcct gctctggtgc ctggcctcgc
     1201 gccgccgtgt atcgccccgc cctgggcggc aaggctggcc cggtcggcac cagttgcgtg
     1261 agcggaaaga tggccgcttc ccggccctgc tgcagggagc tcaaaatgga ggacgcggcg
     1321 ctcgggagag cgggcgggtg agtcacccac acaaaggaaa agggcctttc cgtcctcagc
     1381 cgtcgcttca tgtgactcca cggagtaccg ggcgccgtcc aggcacctcg attagttctc
     1441 gagcttttgg agtacgtcgt ctttaggttg gggggagggg ttttatgcga tggagtttcc
     1501 ccacactgag tgggtggaga ctgaagttag gccagcttgg cacttgatgt aattctcctt
     1561 ggaatttgcc ctttttgagt ttggatcttg gttcattctc aagcctcaga cagtggttca
     1621 aagttttttt cttccatttc aggtgtcgtg aggaattagc ttggtactaa tacgactcac
     1681 tatagggaga cccaagctgg ctaggtaagc ttgatcaaca agtttgtaca aaaaagcagg
     1741 cagctagcgc taccggactc agatccaccg gtcgccacca tggtgagcaa gggcgaggag
     1801 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1861 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1921 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1981 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     2041 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     2101 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     2161 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     2221 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gagtggatcg
     2281 ggtaccgaat tccatgatct tttttttttt tttttttttc ccatgatcat cccggggatc
     2341 ctgcagatgc agtcagattt ctgaactgtc tcagcaaatg gtaagtaact tagagctagt
     2401 aattaataac ctgtcctttg atttctgatt cagccaagaa tggccatatt tgggaaaggc
     2461 agatctggag agtaaccacg ttttcatgca gtctagactg ttaacgcagt gtttctctaa
     2521 ctctcaggtg aacttcaaga tccgccacaa catcgaggac ggcagcgtgc agctcgccga
     2581 ccactaccag cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta
     2641 cctgagcacc cagtccgccc tgagcaaaga ccccaacgag aagcgcgatc acatggtcct
     2701 gctggagttc gtgaccgccg ccgggatcac tctcggcatg gacgagctgt acaagtaaag
     2761 cggccgcgac tctagacgcg tactagtgcc cagctttctt gtacaaagtg gttgatctag
     2821 agggcccgcg gttcgaaggt aagcctatcc ctaaccctct cctcggtctc gattctacgc
     2881 gtaccggtca tcatcaccat caccattgag tttaaacccg ctgatcagcc tcgactgtgc
     2941 cttctagttg ccagccatct gttgtttgcc cctcccccgt gccttccttg accctggaag
     3001 gtgccactcc cactgtcctt tcctaataaa atgaggaaat tgcatcgcat tgtctgagta
     3061 ggtgtcattc tattctgggg ggtggggtgg ggcaggacag caagggggag gattgggaag
     3121 acaatagcag gcatgctggg gatgcggtgg gctctatggc ttctgaggcg gaaagaacca
     3181 gctggggctc tagggggtat ccccacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     3241 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     3301 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     3361 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     3421 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3481 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3541 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3601 atgagctgat ttaacaaaaa tttaacgcga attaattctg tggaatgtgt gtcagttagg
     3661 gtgtggaaag tccccaggct ccccagcagg cagaagtatg caaagcatgc atctcaatta
     3721 gtcagcaacc aggtgtggaa agtccccagg ctccccagca ggcagaagta tgcaaagcat
     3781 gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc cgcccctaac
     3841 tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta tttatgcaga
     3901 ggccgaggcc gcctctgcct ctgagctatt ccagaagtag tgaggaggct tttttggagg
     3961 cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc attttcggat ctgatcagca
     4021 cgtgttgaca attaatcatc ggcatagtat atcggcatag tataatacga caaggtgagg
     4081 aactaaacca tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg
     4141 gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct
     4201 agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca
     4261 gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc
     4321 gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt
     4381 ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca
     4441 gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctaagc acttcgtggc
     4501 cgaggagcag gactgacacg tgctacgaga tttcgattcc accgccgcct tctatgaaag
     4561 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     4621 catgctggag ttcttcgccc accccaactt gtttattgca gcttataatg gttacaaata
     4681 aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg
     4741 tttgtccaaa ctcatcaatg tatcttatca tgtctgtata ccgtcgacct ctagctagag
     4801 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     4861 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     4921 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     4981 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     5041 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     5101 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     5161 gtgagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5221 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5281 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     5341 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     5401 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     5461 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     5521 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     5581 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     5641 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     5701 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     5761 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     5821 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     5881 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     5941 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6001 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6061 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6121 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6181 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6241 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6301 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     6361 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     6421 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     6481 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     6541 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     6601 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     6661 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     6721 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     6781 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
//
