LOCUS       sequence-227459-        4968 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 4968)
  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 4968)
  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 WT
FEATURES             Location/Qualifiers
     source          1..4968
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        104..407
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        408..611
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     562..582
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     regulatory      650..659
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(656..658,659..661,662..1372)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(656..658,659..661,662..1372)
                     /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(701..722)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(962..981)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1309..1330
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1397..1420
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     polyA_signal    1799..1920
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1836..1855)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1890..1909
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     rep_origin      complement(1927..2382)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2064..2085)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     2276..2295
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        2409..2513
                     /gene="bla"
                     /label=AmpR promoter
     promoter        2515..2872
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     enhancer        2515..2706
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     complement(2538..2558)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      2723..2858
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     CDS             2907..3701
                     /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             2907..3701
                     /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(2961..2980)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     3571..3590
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(3889..3908)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    3933..3980
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     rep_origin      4309..4897
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4798..4817
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gttatcccct gattctgtgg ataaccgtat taccgccatg cattagttat taatagtaat
       61 caattacggg gtcattagtt catagcccat atatggagtt ccgcgttaca taacttacgg
      121 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt
      181 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac
      241 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg
      301 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact
      361 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt
      421 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc
      481 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc
      541 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata
      601 taagcagagc tggtttagtg aaccgtcaga tccgctagcg ctaccggtcg ccaccatggt
      661 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
      721 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
      781 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
      841 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
      901 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
      961 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1021 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1081 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     1141 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     1201 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     1261 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     1321 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtccggact
     1381 cagatctcga gagatggact acaaggacga cgatgacaag aattctactg atgtggaaac
     1441 tacgtatgca gattttattg cctcaggaag aacaggtaga agaaatgcaa tacatgatat
     1501 cttggtttcc tctgcaagtg gcaacagcaa tgaattagcc ttgaaattag caggtcttga
     1561 tatcaacaag acagaaggtg aagatgatgg acagagaagc tccacagaac aaagtggcga
     1621 agcccaggga gaagcagcca agtctgaaaa gtagggtacc gcgggcccgg gatccaccgg
     1681 atctagataa ctgatcataa tcagccatac cacatttgta gaggttttac ttgctttaaa
     1741 aaacctccca cacctccccc tgaacctgaa acataaaatg aatgcaattg ttgttgttaa
     1801 cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa
     1861 taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta
     1921 acgcgtaaat tgtaagcgtt aatattttgt taaaattcgc gttaaatttt tgttaaatca
     1981 gctcattttt taaccaatag gccgaaatcg gcaaaatccc ttataaatca aaagaataga
     2041 ccgagatagg gttgagtgtt gttccagttt ggaacaagag tccactatta aagaacgtgg
     2101 actccaacgt caaagggcga aaaaccgtct atcagggcga tggcccacta cgtgaaccat
     2161 caccctaatc aagttttttg gggtcgaggt gccgtaaagc actaaatcgg aaccctaaag
     2221 ggagcccccg atttagagct tgacggggaa agccggcgaa cgtggcgaga aaggaaggga
     2281 agaaagcgaa aggagcgggc gctagggcgc tggcaagtgt agcggtcacg ctgcgcgtaa
     2341 ccaccacacc cgccgcgctt aatgcgccgc tacagggcgc gtcaggtggc acttttcggg
     2401 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     2461 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtcctgagg
     2521 cggaaagaac cagctgtgga atgtgtgtca gttagggtgt ggaaagtccc caggctcccc
     2581 agcaggcaga agtatgcaaa gcatgcatct caattagtca gcaaccaggt gtggaaagtc
     2641 cccaggctcc ccagcaggca gaagtatgca aagcatgcat ctcaattagt cagcaaccat
     2701 agtcccgccc ctaactccgc ccatcccgcc cctaactccg cccagttccg cccattctcc
     2761 gccccatggc tgactaattt tttttattta tgcagaggcc gaggccgcct cggcctctga
     2821 gctattccag aagtagtgag gaggcttttt tggaggccta ggcttttgca aagatcgatc
     2881 aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac gcaggttctc
     2941 cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca atcggctgct
     3001 ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt gtcaagaccg
     3061 acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg tggctggcca
     3121 cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga agggactggc
     3181 tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct cctgccgaga
     3241 aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg gctacctgcc
     3301 cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg gaagccggtc
     3361 ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc gaactgttcg
     3421 ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat ggcgatgcct
     3481 gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac tgtggccggc
     3541 tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt gctgaagagc
     3601 ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct cccgattcgc
     3661 agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc tggggttcga
     3721 aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca ccgccgcctt
     3781 ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga tcctccagcg
     3841 cggggatctc atgctggagt tcttcgccca ccctaggggg aggctaactg aaacacggaa
     3901 ggagacaata ccggaaggaa cccgcgctat gacggcaata aaaagacaga ataaaacgca
     3961 cggtgttggg tcgtttgttc ataaacgcgg ggttcggtcc cagggctggc actctgtcga
     4021 taccccaccg agaccccatt ggggccaata cgcccgcgtt tcttcctttt ccccacccca
     4081 ccccccaagt tcgggtgaag gcccagggct cgcagccaac gtcggggcgg caggccctgc
     4141 catagcctca ggttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     4201 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     4261 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     4321 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     4381 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     4441 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     4501 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     4561 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     4621 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     4681 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     4741 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     4801 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     4861 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     4921 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgc
//
