LOCUS       sequence-327076-        6697 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of EWS-GFP .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6697)
  TITLE     Arrowsmith Lab
REFERENCE   2  (bases 1 to 6697)
  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"
COMMENT     Sequence Label: EWS-GFP
FEATURES             Location/Qualifiers
     source          1..6697
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        54..357
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        358..561
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     512..532
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      621..630
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             2637..3356
                     /codon_start=1
                     /product="Aequorea coerulescens GFP"
                     /label=AcGFP1
                     /note="mammalian codon-optimized"
                     /translation="MVSKGAELFTGIVPILIELNGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLSYGVQCFSRYPDHMKQHDFFKSAMPEGYIQERTIFFEDD
                     GNYKSRAEVKFEGDTLVNRIELTGTDFKEDGNILGNKMEYNYNAHNVYIMTDKAKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMIYF
                     GFVTAAAITHGMDELYK"
     polyA_signal    3478..3599
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3515..3534)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3569..3588
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3606..4061)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3743..3764)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3955..3974
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        4088..4192
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4194..4551
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4194..4385
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4217..4237)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4402..4537
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4586..5380
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             4586..5380
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(4640..4659)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5250..5269
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5568..5587)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5612..5659
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5988..6576
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6477..6496
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 taatagtaat caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca
       61 taacttacgg taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca
      121 ataatgacgt atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg
      181 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      241 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc
      301 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg
      361 atgcggtttt ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca
      421 agtctccacc ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt
      481 ccaaaatgtc gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg
      541 gaggtctata taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggact
      601 cagatctcga gctcaagctt gccaccatgg cgtccacgga ttacagtacc tatagccaag
      661 ctgcagcgca gcagggctac agtgtttaca ccgcccagcc cactcaagga tatgcacgga
      721 ccacccaggc atatgggcaa caaagctatg gaacctatgg acagcccact gatgtcagct
      781 atacccaggc tcagaccact gcaacctatg ggcagaccgc ctatgcaact tcttatggac
      841 agcctcccac tggttatact actccaactg ccccccaggc atacagccag cctgtccagg
      901 ggtatggcac tggtgcttat gataccacca ctgctacagt caccaccacc caggcctcct
      961 atgcagctca gtctgcatat ggcactcagc ctgcttatcc agcctatggg cagcagccag
     1021 cagccactgc acctacaaga ccgcaggatg gaaacaagcc cactgagact agtcaacctc
     1081 aatctagcac agggggttac aaccagccca gcctaggata tggacagagt aactacagtt
     1141 atccccaggt acctgggagc taccccatgc agccagtcac tgcacctcca tcctaccctc
     1201 ctaccagcta ttcctctaca cagccgacta gttatgatca gagcagttac tctcagcaga
     1261 acacctatgg gcaaccgagc agctatggac agcagagtag ctatggtcaa caaagcagct
     1321 atgggcagca gcctcccact agttacccac cccaaactgg atcctacagc caagctccaa
     1381 gtcaatatag ccaacagagc agcagctacg ggcagcagag ttcattccga caggaccacc
     1441 ccagtagcat gggtgtttat gggcaggagt ctggaggatt ttccggacca ggagagaacc
     1501 ggagcatgag tggccctgat aaccggggca ggggaagagg gggatttgat cgtggaggca
     1561 tgagcagagg tgggcgggga ggaggacgcg gtggaatggg cagcgctgga gagcgaggtg
     1621 gcttcaataa gcctggtgga cccatggatg aaggaccaga tcttgatcta ggcccacctg
     1681 tagatccaga tgaagactct gacaacagtg caatttatgt acaaggatta aatgacagtg
     1741 tgactctaga tgatctggca gacttcttta agcagtgtgg ggttgttaag atgaacaaga
     1801 gaactgggca acccatgatc cacatctacc tggacaagga aacaggaaag cccaaaggcg
     1861 atgccacagt gtcctatgaa gacccaccca ctgccaaggc tgccgtggaa tggtttgatg
     1921 ggaaagattt tcaagggagc aaacttaaag tctcccttgc tcggaagaag cctccaatga
     1981 acagtatgcg gggtggtctg ccaccccgtg agggcagagg catgccacca ccactccgtg
     2041 gaggtccagg aggcccagga ggtcctgggg gacccatggg tcgcatggga ggccgtggag
     2101 gagatagagg aggcttccct ccaagaggac cccggggttc ccgagggaac ccctctggag
     2161 gaggaaacgt ccagcaccga gctggagact ggcagtgtcc caatccgggt tgtggaaacc
     2221 agaacttcgc ctggagaaca gagtgcaacc agtgtaaggc cccaaagcct gaaggcttcc
     2281 tcccgccacc ctttccgccc ccgggtggtg atcgtggcag aggtggccct ggtggcatgc
     2341 ggggagaaag aggtggcctc atggatcgtg gtggtcccgg tggaatgttc agaggtggcc
     2401 gtggtggaga cagaggtggc ttccgtggtg gccggggcat ggaccgaggt ggctttggtg
     2461 gaggaagacg aggtggccct ggggggcccc ctggaccttt gatggaacag atgggaggaa
     2521 gaagaggagg acgtggagga cctggaaaaa tggataaagg cgagcaccgt caggagcgca
     2581 gagatcggcc ctactcgaat tctgcagtcg acggtaccgc gggcccggga tccatcatgg
     2641 tgagcaaggg cgccgagctg ttcaccggca tcgtgcccat cctgatcgag ctgaatggcg
     2701 atgtgaatgg ccacaagttc agcgtgagcg gcgagggcga gggcgatgcc acctacggca
     2761 agctgaccct gaagttcatc tgcaccaccg gcaagctgcc tgtgccctgg cccaccctgg
     2821 tgaccaccct gagctacggc gtgcagtgct tctcacgcta ccccgatcac atgaagcagc
     2881 acgacttctt caagagcgcc atgcctgagg gctacatcca ggagcgcacc atcttcttcg
     2941 aggatgacgg caactacaag tcgcgcgccg aggtgaagtt cgagggcgat accctggtga
     3001 atcgcatcga gctgaccggc accgatttca aggaggatgg caacatcctg ggcaataaga
     3061 tggagtacaa ctacaacgcc cacaatgtgt acatcatgac cgacaaggcc aagaatggca
     3121 tcaaggtgaa cttcaagatc cgccacaaca tcgaggatgg cagcgtgcag ctggccgacc
     3181 actaccagca gaataccccc atcggcgatg gccctgtgct gctgcccgat aaccactacc
     3241 tgtccaccca gagcgccctg tccaaggacc ccaacgagaa gcgcgatcac atgatctact
     3301 tcggcttcgt gaccgccgcc gccatcaccc acggcatgga tgagctgtac aagtgagcgg
     3361 ccgcgactct agatcataat cagccatacc acatttgtag aggttttact tgctttaaaa
     3421 aacctcccac acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac
     3481 ttgtttattg cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat
     3541 aaagcatttt tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttaa
     3601 ggcgtaaatt gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag
     3661 ctcatttttt aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac
     3721 cgagataggg ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga
     3781 ctccaacgtc aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc
     3841 accctaatca agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg
     3901 gagcccccga tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa
     3961 gaaagcgaaa ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac
     4021 caccacaccc gccgcgctta atgcgccgct acagggcgcg tcaggtggca cttttcgggg
     4081 aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata tgtatccgct
     4141 catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtcctgaggc
     4201 ggaaagaacc agctgtggaa tgtgtgtcag ttagggtgtg gaaagtcccc aggctcccca
     4261 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccaggtg tggaaagtcc
     4321 ccaggctccc cagcaggcag aagtatgcaa agcatgcatc tcaattagtc agcaaccata
     4381 gtcccgcccc taactccgcc catcccgccc ctaactccgc ccagttccgc ccattctccg
     4441 ccccatggct gactaatttt ttttatttat gcagaggccg aggccgcctc ggcctctgag
     4501 ctattccaga agtagtgagg aggctttttt ggaggcctag gcttttgcaa agatcgatca
     4561 agagacagga tgaggatcgt ttcgcatgat tgaacaagat ggattgcacg caggttctcc
     4621 ggccgcttgg gtggagaggc tattcggcta tgactgggca caacagacaa tcggctgctc
     4681 tgatgccgcc gtgttccggc tgtcagcgca ggggcgcccg gttctttttg tcaagaccga
     4741 cctgtccggt gccctgaatg aactgcaaga cgaggcagcg cggctatcgt ggctggccac
     4801 gacgggcgtt ccttgcgcag ctgtgctcga cgttgtcact gaagcgggaa gggactggct
     4861 gctattgggc gaagtgccgg ggcaggatct cctgtcatct caccttgctc ctgccgagaa
     4921 agtatccatc atggctgatg caatgcggcg gctgcatacg cttgatccgg ctacctgccc
     4981 attcgaccac caagcgaaac atcgcatcga gcgagcacgt actcggatgg aagccggtct
     5041 tgtcgatcag gatgatctgg acgaagagca tcaggggctc gcgccagccg aactgttcgc
     5101 caggctcaag gcgagcatgc ccgacggcga ggatctcgtc gtgacccatg gcgatgcctg
     5161 cttgccgaat atcatggtgg aaaatggccg cttttctgga ttcatcgact gtggccggct
     5221 gggtgtggcg gaccgctatc aggacatagc gttggctacc cgtgatattg ctgaagagct
     5281 tggcggcgaa tgggctgacc gcttcctcgt gctttacggt atcgccgctc ccgattcgca
     5341 gcgcatcgcc ttctatcgcc ttcttgacga gttcttctga gcgggactct ggggttcgaa
     5401 atgaccgacc aagcgacgcc caacctgcca tcacgagatt tcgattccac cgccgccttc
     5461 tatgaaaggt tgggcttcgg aatcgttttc cgggacgccg gctggatgat cctccagcgc
     5521 ggggatctca tgctggagtt cttcgcccac cctaggggga ggctaactga aacacggaag
     5581 gagacaatac cggaaggaac ccgcgctatg acggcaataa aaagacagaa taaaacgcac
     5641 ggtgttgggt cgtttgttca taaacgcggg gttcggtccc agggctggca ctctgtcgat
     5701 accccaccga gaccccattg gggccaatac gcccgcgttt cttccttttc cccaccccac
     5761 cccccaagtt cgggtgaagg cccagggctc gcagccaacg tcggggcggc aggccctgcc
     5821 atagcctcag gttactcata tatactttag attgatttaa aacttcattt ttaatttaaa
     5881 aggatctagg tgaagatcct ttttgataat ctcatgacca aaatccctta acgtgagttt
     5941 tcgttccact gagcgtcaga ccccgtagaa aagatcaaag gatcttcttg agatcctttt
     6001 tttctgcgcg taatctgctg cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt
     6061 ttgccggatc aagagctacc aactcttttt ccgaaggtaa ctggcttcag cagagcgcag
     6121 ataccaaata ctgttcttct agtgtagccg tagttaggcc accacttcaa gaactctgta
     6181 gcaccgccta catacctcgc tctgctaatc ctgttaccag tggctgctgc cagtggcgat
     6241 aagtcgtgtc ttaccgggtt ggactcaaga cgatagttac cggataaggc gcagcggtcg
     6301 ggctgaacgg ggggttcgtg cacacagccc agcttggagc gaacgaccta caccgaactg
     6361 agatacctac agcgtgagct atgagaaagc gccacgcttc ccgaagggag aaaggcggac
     6421 aggtatccgg taagcggcag ggtcggaaca ggagagcgca cgagggagct tccaggggga
     6481 aacgcctggt atctttatag tcctgtcggg tttcgccacc tctgacttga gcgtcgattt
     6541 ttgtgatgct cgtcaggggg gcggagccta tggaaaaacg ccagcaacgc ggccttttta
     6601 cggttcctgg ccttttgctg gccttttgct cacatgttct ttcctgcgtt atcccctgat
     6661 tctgtggata accgtattac cgccatgcat tagttat
//
