LOCUS       sequence-301926-        6462 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6462)
  AUTHORS   Hao JJ, Liu Y, Kruhlak M, Debell KE, Rellahan BL, Shaw S
  TITLE     Phospholipase C-mediated hydrolysis of PIP2 releases ERM proteins 
            from lymphocyte membrane.
  JOURNAL   J Cell Biol. 2009 Feb 9. 184(3):451-62.
  PUBMED    19204146
REFERENCE   2  (bases 1 to 6462)
  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: "J Cell 
            Biol. 2009 Feb 9. 184(3):451-62."
COMMENT     Sequence Label: pHJ322
FEATURES             Location/Qualifiers
     source          1..6462
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..204
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     155..175
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2038..2047
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2044..2046,2047..2049,2050..2763)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2044..2046,2047..2049,2050..2760)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2089..2110)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2350..2369)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2697..2718
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2886..3007
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2923..2942)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2977..2996
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3014..3469)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3151..3172)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3363..3382
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3496..3600
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3602..3959
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3602..3793
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3625..3645)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      3810..3945
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             3994..4788
                     /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             3994..4788
                     /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(4048..4067)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     4658..4677
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(4976..4995)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5020..5067
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5396..5984
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5885..5904
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        6159..6462
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 gtgatgcggt tttggcagta catcaatggg cgtggatagc ggtttgactc acggggattt
       61 ccaagtctcc accccattga cgtcaatggg agtttgtttt ggcaccaaaa tcaacgggac
      121 tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa tgggcggtag gcgtgtacgg
      181 tgggaggtct atataagcag agctggttta gtgaaccgtc agatccgcta gcgctaccgg
      241 actcagatct cgagctcaag cttcgaattc atgcccaaaa cgatcagtgt gcgtgtgacc
      301 accatggatg cagagctgga gtttgccatc cagcccaaca ccaccgggaa gcagctattt
      361 gaccaggtgg tgaaaactat tggcttgagg gaagtttggt tctttggtct gcagtaccag
      421 gacactaaag gtttctccac ctggctgaaa ctcaataaga aggtgactgc ccaggatgtg
      481 cggaaggaaa gccccctgct ctttaagttc cgtgccaagt tctaccctga ggatgtgtcc
      541 gaggaattga ttcaggacat cactcagcgc ctgttctttc tgcaagtgaa agagggcatt
      601 ctcaatgatg atatttactg cccgcctgag accgctgtgc tgctggcctc gtatgctgtc
      661 cagtctaagt atggcgactt caataaggaa gtgcataagt ctggctacct ggccggagac
      721 aagttgctcc cgcagagagt cctggaacag cacaaactca acaaggacca gtgggaggag
      781 cggatccagg tgtggcatga ggaacaccgt ggcatgctca gggaggatgc tgtcctggaa
      841 tatctgaaga ttgctcaaga tctggagatg tatggtgtga actacttcag catcaagaac
      901 aagaaaggct cagagctgtg gctgggggtg gatgccctgg gtctcaacat ctatgagcag
      961 aatgacagac taactcccaa gataggcttc ccctggagtg aaatcaggaa catctctttc
     1021 aatgataaga aatttgtcat caagcccatt gacaaaaaag ccccggactt cgtcttctat
     1081 gctccccggc tgcggattaa caagcggatc ttggccttgt gcatggggaa ccatgaacta
     1141 tacatgcgcc gtcgcaagcc tgataccatt gaggtgcagc agatgaaggc acaggcccgg
     1201 gaggagaagc accagaagca gatggagcgt gctatgctgg aaaatgagaa gaagaagcgt
     1261 gaaatggcag agaaggagaa agagaagatt gaacgggaga aggaggagct gatggagagg
     1321 ctgaagcaga tcgaggaaca gactaagaag gctcagcaag aactggaaga acagacccgt
     1381 agggctctgg aacttgagca ggaacggaag cgtgcccaga gcgaggctga aaagctggcc
     1441 aaggagcgtc aagaagctga agaggccaag gaggccttgc tgcaggcctc ccgggaccag
     1501 aaaaagactc aggaacagct ggccttggaa atggcagagc tgacagctcg aatctcccag
     1561 ctggagatgg cccgacagaa gaaggagagt gaggctgtgg agtggcagca gaaggcccag
     1621 atggtacagg aagacttgga gaagacccgt gctgagctga agactgccat gagtacacct
     1681 catgtggcag agcctgctga gaatgagcag gatgagcagg atgagaatgg ggcagaggct
     1741 agtgctgacc tacgggctga tgctatggcc aaggaccgca gtgaggagga acgtaccact
     1801 gaggcagaga agaatgagcg tgtgcagaag cacctgaagg ccctcacttc ggagctggcc
     1861 aatgccagag atgagtccaa gaagactgcc aatgacatga tccatgctga gaacatgcga
     1921 ctgggccgag acaaatacaa ggacctgcgc cagatccggc agggcaacac caagcagcgc
     1981 attgacgaat ttgagtctat gccgtcgacg gtaccgcggg cccgggatcc accggtcgcc
     2041 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2101 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     2161 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     2221 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     2281 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2341 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2401 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2461 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     2521 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     2581 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     2641 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     2701 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     2761 taaagcggcc gcgactctag atcataatca gccataccac atttgtagag gttttacttg
     2821 ctttaaaaaa cctcccacac ctccccctga acctgaaaca taaaatgaat gcaattgttg
     2881 ttgttaactt gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt
     2941 tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg
     3001 tatcttaagg cgtaaattgt aagcgttaat attttgttaa aattcgcgtt aaatttttgt
     3061 taaatcagct cattttttaa ccaataggcc gaaatcggca aaatccctta taaatcaaaa
     3121 gaatagaccg agatagggtt gagtgttgtt ccagtttgga acaagagtcc actattaaag
     3181 aacgtggact ccaacgtcaa agggcgaaaa accgtctatc agggcgatgg cccactacgt
     3241 gaaccatcac cctaatcaag ttttttgggg tcgaggtgcc gtaaagcact aaatcggaac
     3301 cctaaaggga gcccccgatt tagagcttga cggggaaagc cggcgaacgt ggcgagaaag
     3361 gaagggaaga aagcgaaagg agcgggcgct agggcgctgg caagtgtagc ggtcacgctg
     3421 cgcgtaacca ccacacccgc cgcgcttaat gcgccgctac agggcgcgtc aggtggcact
     3481 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3541 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3601 cctgaggcgg aaagaaccag ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag
     3661 gctccccagc aggcagaagt atgcaaagca tgcatctcaa ttagtcagca accaggtgtg
     3721 gaaagtcccc aggctcccca gcaggcagaa gtatgcaaag catgcatctc aattagtcag
     3781 caaccatagt cccgccccta actccgccca tcccgcccct aactccgccc agttccgccc
     3841 attctccgcc ccatggctga ctaatttttt ttatttatgc agaggccgag gccgcctcgg
     3901 cctctgagct attccagaag tagtgaggag gcttttttgg aggcctaggc ttttgcaaag
     3961 atcgatcaag agacaggatg aggatcgttt cgcatgattg aacaagatgg attgcacgca
     4021 ggttctccgg ccgcttgggt ggagaggcta ttcggctatg actgggcaca acagacaatc
     4081 ggctgctctg atgccgccgt gttccggctg tcagcgcagg ggcgcccggt tctttttgtc
     4141 aagaccgacc tgtccggtgc cctgaatgaa ctgcaagacg aggcagcgcg gctatcgtgg
     4201 ctggccacga cgggcgttcc ttgcgcagct gtgctcgacg ttgtcactga agcgggaagg
     4261 gactggctgc tattgggcga agtgccgggg caggatctcc tgtcatctca ccttgctcct
     4321 gccgagaaag tatccatcat ggctgatgca atgcggcggc tgcatacgct tgatccggct
     4381 acctgcccat tcgaccacca agcgaaacat cgcatcgagc gagcacgtac tcggatggaa
     4441 gccggtcttg tcgatcagga tgatctggac gaagagcatc aggggctcgc gccagccgaa
     4501 ctgttcgcca ggctcaaggc gagcatgccc gacggcgagg atctcgtcgt gacccatggc
     4561 gatgcctgct tgccgaatat catggtggaa aatggccgct tttctggatt catcgactgt
     4621 ggccggctgg gtgtggcgga ccgctatcag gacatagcgt tggctacccg tgatattgct
     4681 gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc tttacggtat cgccgctccc
     4741 gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt tcttctgagc gggactctgg
     4801 ggttcgaaat gaccgaccaa gcgacgccca acctgccatc acgagatttc gattccaccg
     4861 ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg ggacgccggc tggatgatcc
     4921 tccagcgcgg ggatctcatg ctggagttct tcgcccaccc tagggggagg ctaactgaaa
     4981 cacggaagga gacaataccg gaaggaaccc gcgctatgac ggcaataaaa agacagaata
     5041 aaacgcacgg tgttgggtcg tttgttcata aacgcggggt tcggtcccag ggctggcact
     5101 ctgtcgatac cccaccgaga ccccattggg gccaatacgc ccgcgtttct tccttttccc
     5161 caccccaccc cccaagttcg ggtgaaggcc cagggctcgc agccaacgtc ggggcggcag
     5221 gccctgccat agcctcaggt tactcatata tactttagat tgatttaaaa cttcattttt
     5281 aatttaaaag gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac
     5341 gtgagttttc gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag
     5401 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     5461 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     5521 gagcgcagat accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga
     5581 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     5641 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5701 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5761 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5821 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5881 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5941 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     6001 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     6061 cccctgattc tgtggataac cgtattaccg ccatgcatta gttattaata gtaatcaatt
     6121 acggggtcat tagttcatag cccatatatg gagttccgcg ttacataact tacggtaaat
     6181 ggcccgcctg gctgaccgcc caacgacccc cgcccattga cgtcaataat gacgtatgtt
     6241 cccatagtaa cgccaatagg gactttccat tgacgtcaat gggtggagta tttacggtaa
     6301 actgcccact tggcagtaca tcaagtgtat catatgccaa gtacgccccc tattgacgtc
     6361 aatgacggta aatggcccgc ctggcattat gcccagtaca tgaccttatg ggactttcct
     6421 acttggcagt acatctacgt attagtcatc gctattacca tg
//
