LOCUS       sequence-156371-        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: pHJ320
FEATURES             Location/Qualifiers
     source          1..6462
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        68..371
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        372..575
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     526..546
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      2409..2418
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2415..2417,2418..2420,2421..3134)
                     /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(2415..2417,2418..2420,2421..3131)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2460..2481)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2721..2740)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3068..3089
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3257..3378
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3294..3313)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3348..3367
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3385..3840)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3522..3543)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3734..3753
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3867..3971
                     /gene="bla"
                     /label=AmpR promoter
     promoter        3973..4330
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        3973..4164
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(3996..4016)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4181..4316
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4365..5159
                     /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             4365..5159
                     /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(4419..4438)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5029..5048
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5347..5366)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5391..5438
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5767..6355
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6256..6275
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 catgcattag ttattaatag taatcaatta cggggtcatt agttcatagc ccatatatgg
       61 agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc aacgaccccc
      121 gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg actttccatt
      181 gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat caagtgtatc
      241 atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc tggcattatg
      301 cccagtacat gaccttatgg gactttccta cttggcagta catctacgta ttagtcatcg
      361 ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag cggtttgact
      421 cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt tggcaccaaa
      481 atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa atgggcggta
      541 ggcgtgtacg gtgggaggtc tatataagca gagctggttt agtgaaccgt cagatccgct
      601 agcgctaccg gactcagatc tcgagctcaa gcttcgaatt catgcccaaa acgatcagtg
      661 tgcgtgtgac caccatggat gcagagctgg agtttgccat ccagcccaac accaccggga
      721 agcagctatt tgaccaggtg gtgaaaacta ttggcttgag ggaagtttgg ttctttggtc
      781 tgcagtacca ggacactaaa ggtttctcca cctggctgaa actcaataag aaggtgactg
      841 cccaggatgt gcggaaggaa agccccctgc tctttaagtt ccgtgccaag ttctaccctg
      901 aggatgtgtc cgaggaattg attcaggaca tcactcagcg cctgttcttt ctgcaagtga
      961 aagagggcat tctcaatgat gatatttact gcccgcctga gaccgctgtg ctgctggcct
     1021 cgtatgctgt ccagtctaag tatggcgact tcaataagga agtgcataag tctggctacc
     1081 tggccggaga caagttgctc ccgcagagag tcctggaaca gcacaaactc aacaaggacc
     1141 agtgggagga gcggatccag gtgtggcatg aggaacaccg tggcatgctc agggaggatg
     1201 ctgtcctgga atatctgaag attgctcaag atctggagat gtatggtgtg aactacttca
     1261 gcatcaagaa caagaaaggc tcagagctgt ggctgggggt ggatgccctg ggtctcaaca
     1321 tctatgagca gaatgacaga ctaactccca agataggctt cccctggagt gaaatcagga
     1381 acatctcttt caatgataag aaatttgtca tcaagcccat tgacaaaaaa gccccggact
     1441 tcgtcttcta tgctccccgg ctgcggatta acaagcggat cttggccttg tgcatgggga
     1501 accatgaact atacatgcgc cgtcgcaagc ctgataccat tgaggtgcag cagatgaagg
     1561 cacaggcccg ggaggagaag caccagaagc agatggagcg tgctatgctg gaaaatgaga
     1621 agaagaagcg tgaaatggca gagaaggaga aagagaagat tgaacgggag aaggaggagc
     1681 tgatggagag gctgaagcag atcgaggaac agactaagaa ggctcagcaa gaactggaag
     1741 aacagacccg tagggctctg gaacttgagc aggaacggaa gcgtgcccag agcgaggctg
     1801 aaaagctggc caaggagcgt caagaagctg aagaggccaa ggaggccttg ctgcaggcct
     1861 cccgggacca gaaaaagact caggaacagc tggccttgga aatggcagag ctgacagctc
     1921 gaatctccca gctggagatg gcccgacaga agaaggagag tgaggctgtg gagtggcagc
     1981 agaaggccca gatggtacag gaagacttgg agaagacccg tgctgagctg aagactgcca
     2041 tgagtacacc tcatgtggca gagcctgctg agaatgagca ggatgagcag gatgagaatg
     2101 gggcagaggc tagtgctgac ctacgggctg atgctatggc caaggaccgc agtgaggagg
     2161 aacgtaccac tgaggcagag aagaatgagc gtgtgcagaa gcacctgaag gccctcactt
     2221 cggagctggc caatgccaga gatgagtcca agaagactgc caatgacatg atccatgctg
     2281 agaacatgcg actgggccga gacaaataca agaccctgcg ccagatccgg cagggcaaca
     2341 ccaagcagcg cattgacgaa tttgagtcta tgccgtcgac ggtaccgcgg gcccgggatc
     2401 caccggtcgc caccatggtg agcaagggcg aggagctgtt caccggggtg gtgcccatcc
     2461 tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtgtccggc gagggcgagg
     2521 gcgatgccac ctacggcaag ctgaccctga agttcatctg caccaccggc aagctgcccg
     2581 tgccctggcc caccctcgtg accaccctga cctacggcgt gcagtgcttc agccgctacc
     2641 ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc tacgtccagg
     2701 agcgcaccat cttcttcaag gacgacggca actacaagac ccgcgccgag gtgaagttcg
     2761 agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag gaggacggca
     2821 acatcctggg gcacaagctg gagtacaact acaacagcca caacgtctat atcatggccg
     2881 acaagcagaa gaacggcatc aaggtgaact tcaagatccg ccacaacatc gaggacggca
     2941 gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc cccgtgctgc
     3001 tgcccgacaa ccactacctg agcacccagt ccgccctgag caaagacccc aacgagaagc
     3061 gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gatcactctc ggcatggacg
     3121 agctgtacaa gtaaagcggc cgcgactcta gatcataatc agccatacca catttgtaga
     3181 ggttttactt gctttaaaaa acctcccaca cctccccctg aacctgaaac ataaaatgaa
     3241 tgcaattgtt gttgttaact tgtttattgc agcttataat ggttacaaat aaagcaatag
     3301 catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg gtttgtccaa
     3361 actcatcaat gtatcttaag gcgtaaattg taagcgttaa tattttgtta aaattcgcgt
     3421 taaatttttg ttaaatcagc tcatttttta accaataggc cgaaatcggc aaaatccctt
     3481 ataaatcaaa agaatagacc gagatagggt tgagtgttgt tccagtttgg aacaagagtc
     3541 cactattaaa gaacgtggac tccaacgtca aagggcgaaa aaccgtctat cagggcgatg
     3601 gcccactacg tgaaccatca ccctaatcaa gttttttggg gtcgaggtgc cgtaaagcac
     3661 taaatcggaa ccctaaaggg agcccccgat ttagagcttg acggggaaag ccggcgaacg
     3721 tggcgagaaa ggaagggaag aaagcgaaag gagcgggcgc tagggcgctg gcaagtgtag
     3781 cggtcacgct gcgcgtaacc accacacccg ccgcgcttaa tgcgccgcta cagggcgcgt
     3841 caggtggcac ttttcgggga aatgtgcgcg gaacccctat ttgtttattt ttctaaatac
     3901 attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa taatattgaa
     3961 aaaggaagag tcctgaggcg gaaagaacca gctgtggaat gtgtgtcagt tagggtgtgg
     4021 aaagtcccca ggctccccag caggcagaag tatgcaaagc atgcatctca attagtcagc
     4081 aaccaggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct
     4141 caattagtca gcaaccatag tcccgcccct aactccgccc atcccgcccc taactccgcc
     4201 cagttccgcc cattctccgc cccatggctg actaattttt tttatttatg cagaggccga
     4261 ggccgcctcg gcctctgagc tattccagaa gtagtgagga ggcttttttg gaggcctagg
     4321 cttttgcaaa gatcgatcaa gagacaggat gaggatcgtt tcgcatgatt gaacaagatg
     4381 gattgcacgc aggttctccg gccgcttggg tggagaggct attcggctat gactgggcac
     4441 aacagacaat cggctgctct gatgccgccg tgttccggct gtcagcgcag gggcgcccgg
     4501 ttctttttgt caagaccgac ctgtccggtg ccctgaatga actgcaagac gaggcagcgc
     4561 ggctatcgtg gctggccacg acgggcgttc cttgcgcagc tgtgctcgac gttgtcactg
     4621 aagcgggaag ggactggctg ctattgggcg aagtgccggg gcaggatctc ctgtcatctc
     4681 accttgctcc tgccgagaaa gtatccatca tggctgatgc aatgcggcgg ctgcatacgc
     4741 ttgatccggc tacctgccca ttcgaccacc aagcgaaaca tcgcatcgag cgagcacgta
     4801 ctcggatgga agccggtctt gtcgatcagg atgatctgga cgaagagcat caggggctcg
     4861 cgccagccga actgttcgcc aggctcaagg cgagcatgcc cgacggcgag gatctcgtcg
     4921 tgacccatgg cgatgcctgc ttgccgaata tcatggtgga aaatggccgc ttttctggat
     4981 tcatcgactg tggccggctg ggtgtggcgg accgctatca ggacatagcg ttggctaccc
     5041 gtgatattgc tgaagagctt ggcggcgaat gggctgaccg cttcctcgtg ctttacggta
     5101 tcgccgctcc cgattcgcag cgcatcgcct tctatcgcct tcttgacgag ttcttctgag
     5161 cgggactctg gggttcgaaa tgaccgacca agcgacgccc aacctgccat cacgagattt
     5221 cgattccacc gccgccttct atgaaaggtt gggcttcgga atcgttttcc gggacgccgg
     5281 ctggatgatc ctccagcgcg gggatctcat gctggagttc ttcgcccacc ctagggggag
     5341 gctaactgaa acacggaagg agacaatacc ggaaggaacc cgcgctatga cggcaataaa
     5401 aagacagaat aaaacgcacg gtgttgggtc gtttgttcat aaacgcgggg ttcggtccca
     5461 gggctggcac tctgtcgata ccccaccgag accccattgg ggccaatacg cccgcgtttc
     5521 ttccttttcc ccaccccacc ccccaagttc gggtgaaggc ccagggctcg cagccaacgt
     5581 cggggcggca ggccctgcca tagcctcagg ttactcatat atactttaga ttgatttaaa
     5641 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     5701 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     5761 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     5821 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     5881 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     5941 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     6001 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     6061 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     6121 aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc
     6181 cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac
     6241 gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct
     6301 ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc
     6361 cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgttctt
     6421 tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gc
//
