LOCUS       sequence-142584-        6495 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 6495)
  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 6495)
  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: pHJ423
FEATURES             Location/Qualifiers
     source          1..6495
                     /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      2442..2451
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2448..2450,2451..2453,2454..3167)
                     /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(2448..2450,2451..2453,2454..3164)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2493..2514)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2754..2773)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3101..3122
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    3290..3411
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3327..3346)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     3381..3400
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(3418..3873)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3555..3576)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3767..3786
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        3900..4004
                     /gene="bla"
                     /label=AmpR promoter
     promoter        4006..4363
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        4006..4197
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(4029..4049)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      4214..4349
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             4398..5192
                     /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             4398..5192
                     /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(4452..4471)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     5062..5081
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(5380..5399)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    5424..5471
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      5800..6388
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6289..6308
                     /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 cccgaaaatg ccgaaaccaa
      661 tcaatgtccg agttaccacc atggatgcag agctggagtt tgcaatccag ccaaatacaa
      721 ctggaaaaca gctttttgat caggtggtaa agactatcgg cctccgggaa gtgtggtact
      781 ttggcctcca ctatgtggat aataaaggat ttcctacctg gctgaagctg gataagaagg
      841 tgtctgccca ggaggtcagg aaggagaatc ccctccagtt caagttccgg gccaagttct
      901 accctgaaga tgtggctgag gagctcatcc aggacatcac ccagaaactt ttcttcctcc
      961 aagtgaagga aggaatcctt agcgatgaga tctactgccc ccctgagact gccgtgctct
     1021 tggggtccta cgctgtgcag gccaagtttg gggactacaa caaagaagtg cacaagtctg
     1081 ggtacctcag ctctgagcgg ctgatccctc aaagagtgat ggaccagcac aaacttacca
     1141 gggaccagtg ggaggaccgg atccaggtgt ggcatgcgga acaccgtggg atgctcaaag
     1201 ataatgctat gttggaatac ctgaagattg ctcaggacct ggaaatgtat ggaatcaact
     1261 atttcgagat aaaaaacaag aaaggaacag acctttggct tggagttgat gcccttggac
     1321 tgaatattta tgagaaagat gataagttaa ccccaaagat tggctttcct tggagtgaaa
     1381 tcaggaacat ctctttcaat gacaaaaagt ttgtcattaa acccatcgac aagaaggcac
     1441 ctgactttgt gttttatgcc ccacgtctga gaatcaacaa gcggatcctg cagctctgca
     1501 tgggcaacca tgagttgtat atgcgccgca ggaagcctga caccatcgag gtgcagcaga
     1561 tgaaggccca ggcccgggag gagaagcatc agaagcagct ggagcggcaa cagctggaaa
     1621 cagagaagaa aaggagagaa accgtggaga gagagaaaga gcagatgatg cgcgagaagg
     1681 aggagttgat gctgcggctg caggactatg aggagaagac aaagaaggca gagagagagc
     1741 tctcggagca gattcagagg gccctgcagc tggaggagga gaggaagcgg gcacaggagg
     1801 aggccgagcg cctagaggct gaccgtatgg ctgcactgcg ggctaaggag gagctggaga
     1861 gacaggcggt ggatcagata aagagccagg agcagctggc tgcggagctt gcagaataca
     1921 ctgccaagat tgccctcctg gaagaggcgc ggaggcgcaa ggaggatgaa gttgaagagt
     1981 ggcagcacag ggccaaagaa gcccaggatg acctggtgaa gaccaaggag gagctgcacc
     2041 tggtgatgac agcacccccg cccccaccac cccccgtgta cgagccggtg agctaccatg
     2101 tccaggagag cttgcaggat gagggcgcag agcccacggg ctacagcgcg gagctgtcta
     2161 gtgagggcat ccgggatgac cgcaatgagg agaagcgcat cactgaggca gagaagaacg
     2221 agcgtgtgca gcggcagctg ctgacgctga gcagcgagct gtcccaggcc cgagatgaga
     2281 ataagaggac ccacaatgac atcatccaca acgagaacat gaggcaaggc cgggacaagt
     2341 acaaggacct gcggcagatc cggcagggca acaccaagca gcgcatcgac gagttcgagg
     2401 ccctgtcgtc gacggtaccg cgggcccggg atccaccggt cgccaccatg gtgagcaagg
     2461 gcgaggagct gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg
     2521 gccacaagtt cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc
     2581 tgaagttcat ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc
     2641 tgacctacgg cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct
     2701 tcaagtccgc catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg
     2761 gcaactacaa gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg
     2821 agctgaaggg catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca
     2881 actacaacag ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga
     2941 acttcaagat ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc
     3001 agaacacccc catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc
     3061 agtccgccct gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg
     3121 tgaccgccgc cgggatcact ctcggcatgg acgagctgta caagtaaagc ggccgcgact
     3181 ctagatcata atcagccata ccacatttgt agaggtttta cttgctttaa aaaacctccc
     3241 acacctcccc ctgaacctga aacataaaat gaatgcaatt gttgttgtta acttgtttat
     3301 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt
     3361 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatctt aaggcgtaaa
     3421 ttgtaagcgt taatattttg ttaaaattcg cgttaaattt ttgttaaatc agctcatttt
     3481 ttaaccaata ggccgaaatc ggcaaaatcc cttataaatc aaaagaatag accgagatag
     3541 ggttgagtgt tgttccagtt tggaacaaga gtccactatt aaagaacgtg gactccaacg
     3601 tcaaagggcg aaaaaccgtc tatcagggcg atggcccact acgtgaacca tcaccctaat
     3661 caagtttttt ggggtcgagg tgccgtaaag cactaaatcg gaaccctaaa gggagccccc
     3721 gatttagagc ttgacgggga aagccggcga acgtggcgag aaaggaaggg aagaaagcga
     3781 aaggagcggg cgctagggcg ctggcaagtg tagcggtcac gctgcgcgta accaccacac
     3841 ccgccgcgct taatgcgccg ctacagggcg cgtcaggtgg cacttttcgg ggaaatgtgc
     3901 gcggaacccc tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac
     3961 aataaccctg ataaatgctt caataatatt gaaaaaggaa gagtcctgag gcggaaagaa
     4021 ccagctgtgg aatgtgtgtc agttagggtg tggaaagtcc ccaggctccc cagcaggcag
     4081 aagtatgcaa agcatgcatc tcaattagtc agcaaccagg tgtggaaagt ccccaggctc
     4141 cccagcaggc agaagtatgc aaagcatgca tctcaattag tcagcaacca tagtcccgcc
     4201 cctaactccg cccatcccgc ccctaactcc gcccagttcc gcccattctc cgccccatgg
     4261 ctgactaatt ttttttattt atgcagaggc cgaggccgcc tcggcctctg agctattcca
     4321 gaagtagtga ggaggctttt ttggaggcct aggcttttgc aaagatcgat caagagacag
     4381 gatgaggatc gtttcgcatg attgaacaag atggattgca cgcaggttct ccggccgctt
     4441 gggtggagag gctattcggc tatgactggg cacaacagac aatcggctgc tctgatgccg
     4501 ccgtgttccg gctgtcagcg caggggcgcc cggttctttt tgtcaagacc gacctgtccg
     4561 gtgccctgaa tgaactgcaa gacgaggcag cgcggctatc gtggctggcc acgacgggcg
     4621 ttccttgcgc agctgtgctc gacgttgtca ctgaagcggg aagggactgg ctgctattgg
     4681 gcgaagtgcc ggggcaggat ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca
     4741 tcatggctga tgcaatgcgg cggctgcata cgcttgatcc ggctacctgc ccattcgacc
     4801 accaagcgaa acatcgcatc gagcgagcac gtactcggat ggaagccggt cttgtcgatc
     4861 aggatgatct ggacgaagag catcaggggc tcgcgccagc cgaactgttc gccaggctca
     4921 aggcgagcat gcccgacggc gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga
     4981 atatcatggt ggaaaatggc cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg
     5041 cggaccgcta tcaggacata gcgttggcta cccgtgatat tgctgaagag cttggcggcg
     5101 aatgggctga ccgcttcctc gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg
     5161 ccttctatcg ccttcttgac gagttcttct gagcgggact ctggggttcg aaatgaccga
     5221 ccaagcgacg cccaacctgc catcacgaga tttcgattcc accgccgcct tctatgaaag
     5281 gttgggcttc ggaatcgttt tccgggacgc cggctggatg atcctccagc gcggggatct
     5341 catgctggag ttcttcgccc accctagggg gaggctaact gaaacacgga aggagacaat
     5401 accggaagga acccgcgcta tgacggcaat aaaaagacag aataaaacgc acggtgttgg
     5461 gtcgtttgtt cataaacgcg gggttcggtc ccagggctgg cactctgtcg ataccccacc
     5521 gagaccccat tggggccaat acgcccgcgt ttcttccttt tccccacccc accccccaag
     5581 ttcgggtgaa ggcccagggc tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc
     5641 aggttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     5701 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     5761 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5821 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5881 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5941 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     6001 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     6061 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     6121 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     6181 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     6241 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6301 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     6361 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     6421 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     6481 taaccgtatt accgc
//
