LOCUS       sequence-227476-        4920 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expression of PKIa.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4920)
  AUTHORS   Billiard J, Grewal SS, Lukaesko L, Stork PJ, Rotwein P
  TITLE     Hormonal control of insulin-like growth factor I gene transcription 
            in human osteoblasts: dual actions of cAMP-dependent protein kinase 
            on CCAAT/enhancer-binding protein delta.
  JOURNAL   J Biol Chem. 2001 Aug 17;276(33):31238-46. doi: 
            10.1074/jbc.M103634200. Epub 2001 Jun 4.
  PUBMED    11390399
REFERENCE   2  (bases 1 to 4920)
  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"; doi: 
            "10.1074/jbc.M103634200"; journalName: "J Biol Chem"; date: 
            "2001-08-17- 17"; volume: "276"; issue: "33"; pages: "31238-46"
COMMENT     Sequence Label: GFP-PKI nls
FEATURES             Location/Qualifiers
     source          1..4920
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        125..428
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        429..632
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     583..603
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      671..680
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(677..679,680..682,683..1393)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(677..679,680..682,683..1393)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(722..743)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(983..1002)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1330..1351
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1418..1465
                     /codon_start=1
                     /product="two tandem FLAG(R) epitope tags"
                     /label=2xFLAG
                     /translation="DYKDDDDKDYKDDDDK"
     CDS             1580..1600
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             1622..1642
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     polyA_signal    1772..1893
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1809..1828)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1863..1882
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1900..2355)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2037..2058)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2249..2268
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2382..2486
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2488..2845
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2488..2679
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2511..2531)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2696..2831
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2880..3674
                     /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             2880..3674
                     /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(2934..2953)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3544..3563
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3862..3881)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3906..3953
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4282..4870
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4771..4790
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tgctcacatg ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgccat
       61 gcattagtta ttaatagtaa tcaattacgg ggtcattagt tcatagccca tatatggagt
      121 tccgcgttac ataacttacg gtaaatggcc cgcctggctg accgcccaac gacccccgcc
      181 cattgacgtc aataatgacg tatgttccca tagtaacgcc aatagggact ttccattgac
      241 gtcaatgggt ggagtattta cggtaaactg cccacttggc agtacatcaa gtgtatcata
      301 tgccaagtac gccccctatt gacgtcaatg acggtaaatg gcccgcctgg cattatgccc
      361 agtacatgac cttatgggac tttcctactt ggcagtacat ctacgtatta gtcatcgcta
      421 ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagcgg tttgactcac
      481 ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg caccaaaatc
      541 aacgggactt tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg ggcggtaggc
      601 gtgtacggtg ggaggtctat ataagcagag ctggtttagt gaaccgtcag atccgctagc
      661 gctaccggtc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
      721 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg gcgagggcga
      781 gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
      841 cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct tcagccgcta
      901 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
      961 ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg aggtgaagtt
     1021 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1081 caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct atatcatggc
     1141 cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca tcgaggacgg
     1201 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1261 gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc ccaacgagaa
     1321 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggatcactc tcggcatgga
     1381 cgagctgtac aagtccggac tcagatctcg agagatggac tacaaggacg acgatgacaa
     1441 ggactacaag gacgacgatg acaagaattc tactgatgtg gaaactacgt atgcagattt
     1501 tattgcctca ggaagaacag gtagaagaaa tgcaatacat gatatcttgg tttcctctgc
     1561 aagtggcaac agcaatgaac ctaagaagaa acgcaaggtc ggtaccggag gaggtgaagc
     1621 acccaagaaa aagcgaaaag taggatccac cggatctaga taactgatca taatcagcca
     1681 taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc ccctgaacct
     1741 gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt ataatggtta
     1801 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     1861 ttgtggtttg tccaaactca tcaatgtatc ttaacgcgta aattgtaagc gttaatattt
     1921 tgttaaaatt cgcgttaaat ttttgttaaa tcagctcatt ttttaaccaa taggccgaaa
     1981 tcggcaaaat cccttataaa tcaaaagaat agaccgagat agggttgagt gttgttccag
     2041 tttggaacaa gagtccacta ttaaagaacg tggactccaa cgtcaaaggg cgaaaaaccg
     2101 tctatcaggg cgatggccca ctacgtgaac catcacccta atcaagtttt ttggggtcga
     2161 ggtgccgtaa agcactaaat cggaacccta aagggagccc ccgatttaga gcttgacggg
     2221 gaaagccggc gaacgtggcg agaaaggaag ggaagaaagc gaaaggagcg ggcgctaggg
     2281 cgctggcaag tgtagcggtc acgctgcgcg taaccaccac acccgccgcg cttaatgcgc
     2341 cgctacaggg cgcgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt
     2401 tatttttcta aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc
     2461 ttcaataata ttgaaaaagg aagagtcctg aggcggaaag aaccagctgt ggaatgtgtg
     2521 tcagttaggg tgtggaaagt ccccaggctc cccagcaggc agaagtatgc aaagcatgca
     2581 tctcaattag tcagcaacca ggtgtggaaa gtccccaggc tccccagcag gcagaagtat
     2641 gcaaagcatg catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc
     2701 gcccctaact ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat
     2761 ttatgcagag gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt
     2821 ttttggaggc ctaggctttt gcaaagatcg atcaagagac aggatgagga tcgtttcgca
     2881 tgattgaaca agatggattg cacgcaggtt ctccggccgc ttgggtggag aggctattcg
     2941 gctatgactg ggcacaacag acaatcggct gctctgatgc cgccgtgttc cggctgtcag
     3001 cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc cggtgccctg aatgaactgc
     3061 aagacgaggc agcgcggcta tcgtggctgg ccacgacggg cgttccttgc gcagctgtgc
     3121 tcgacgttgt cactgaagcg ggaagggact ggctgctatt gggcgaagtg ccggggcagg
     3181 atctcctgtc atctcacctt gctcctgccg agaaagtatc catcatggct gatgcaatgc
     3241 ggcggctgca tacgcttgat ccggctacct gcccattcga ccaccaagcg aaacatcgca
     3301 tcgagcgagc acgtactcgg atggaagccg gtcttgtcga tcaggatgat ctggacgaag
     3361 agcatcaggg gctcgcgcca gccgaactgt tcgccaggct caaggcgagc atgcccgacg
     3421 gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc gaatatcatg gtggaaaatg
     3481 gccgcttttc tggattcatc gactgtggcc ggctgggtgt ggcggaccgc tatcaggaca
     3541 tagcgttggc tacccgtgat attgctgaag agcttggcgg cgaatgggct gaccgcttcc
     3601 tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat cgccttctat cgccttcttg
     3661 acgagttctt ctgagcggga ctctggggtt cgaaatgacc gaccaagcga cgcccaacct
     3721 gccatcacga gatttcgatt ccaccgccgc cttctatgaa aggttgggct tcggaatcgt
     3781 tttccgggac gccggctgga tgatcctcca gcgcggggat ctcatgctgg agttcttcgc
     3841 ccaccctagg gggaggctaa ctgaaacacg gaaggagaca ataccggaag gaacccgcgc
     3901 tatgacggca ataaaaagac agaataaaac gcacggtgtt gggtcgtttg ttcataaacg
     3961 cggggttcgg tcccagggct ggcactctgt cgatacccca ccgagacccc attggggcca
     4021 atacgcccgc gtttcttcct tttccccacc ccacccccca agttcgggtg aaggcccagg
     4081 gctcgcagcc aacgtcgggg cggcaggccc tgccatagcc tcaggttact catatatact
     4141 ttagattgat ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga
     4201 taatctcatg accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt
     4261 agaaaagatc aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca
     4321 aacaaaaaaa ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct
     4381 ttttccgaag gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta
     4441 gccgtagtta ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct
     4501 aatcctgtta ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc
     4561 aagacgatag ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca
     4621 gcccagcttg gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga
     4681 aagcgccacg cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg
     4741 aacaggagag cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt
     4801 cgggtttcgc cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag
     4861 cctatggaaa aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt
//
