LOCUS       sequence-370308-        6839 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  One in a series of plasmids to titrate levels of expressed protein 
            to levels of endogenous protein (non-attenuated EF1a promoter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6839)
  AUTHORS   Millette MM, Holland ED, Tenpas TJ, Dent EW
  TITLE     A Single Transcript Knockdown-Replacement Strategy Employing 5' UTR 
            Secondary Structures to Precisely Titrate Rescue Protein 
            Translation.
  JOURNAL   Front Genome Ed. 2022 Mar 28;4:803375. doi: 
            10.3389/fgeed.2022.803375. eCollection 2022.
  PUBMED    35419562
REFERENCE   2  (bases 1 to 6839)
  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: "Front 
            Genome Ed."; date: "2022-03-28"; volume: "4:803375. doi"; pages: " 
            10.3389/fgeed.2022.803375. eCollection 2022"
COMMENT     Sequence Label: pKDR-EF1a
FEATURES             Location/Qualifiers
     source          1..6839
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        36..1218
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          267..1209
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1166..1186
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     regulatory      1239..1248
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1245..1247,1248..1250,1251..1964)
                     /codon_start=1
                     /product="bright green-yellow fluorescent protein derived 
                     from mClover2, with improved photostability (Bajar et al., 
                     2003)"
                     /label=mClover3
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTL
                     KFICTTGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISFKDD
                     GTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYITADKQKNCIK
                     ANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSHQSKLSKDPNEKRDHMVLL
                     EFVTAAGITHGMDELYK"
     primer_bind     complement(1290..1311)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1898..1919
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(2024..2598,2599..2601,2602..2610)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2191..2208)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2418..2437
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     ncRNA           3541..3555
                     /label=miR-30a loop
                     /note="loop from the the 71-nt precursor of the human 
                     miR-30a microRNA (Zeng et al., 2002)"
                     /ncRNA_class="miRNA"
     primer_bind     complement(3861..3880)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3926..3981
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3980..3999)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4342..4358)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4342..4358)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4355..4377)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4366..4382
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     promoter        complement(join(4390..4396,4397..4414,4415..4420))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4435..4456
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4515..4710
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4561..4696
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4716..4850
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4753..4772)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4807..4826
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4918..4935)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5089..5677)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5169..5188)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5848..6639,6640..6708))
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     6471..6490
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6709..6813)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ggtcgacatt gattattgac tagatcatcg cgtgaggctc cggtgcccgt cagtgggcag
       61 agcgcacatc gcccacagtc cccgagaagt tggggggagg ggtcggcaat tgaagcggtg
      121 cctagagaag gtggcgcggg gtaaactggg aaagtgatgt cgtgtactgg ctccgccttt
      181 ttcccgaggg tgggggagaa ccgtatataa gtgcagtagt cgccgtgaac gttctttttc
      241 gcaacgggtt tgccgccaga acacaggtaa gtgccgtgtg tggttcccgc gggcctggcc
      301 tctttacggg ttatggccct tgcgtgcctt gaattacttc cacgcccctg gctgcagtac
      361 gtgattcttg atcccgagct tcgggttgga agtgggtggg agagttcgag gccttgcgct
      421 taaggagccc cttcgcctcg tgcttgagtt gaggcctggc ctgggcgctg gggccgccgc
      481 gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt tcgataagtc tctagccatt
      541 taaaattttt gatgacctgc tgcgacgctt tttttctggc aagatagtct tgtaaatgcg
      601 ggccaagatc tgcacactgg tatttcggtt tttggggccg cgggcggcga cggggcccgt
      661 gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag cgcggccacc gagaatcgga
      721 cgggggtagt ctcaagctgg ccggcctgct ctggtgcctg gcctcgcgcc gccgtgtatc
      781 gccccgccct gggcggcaag gctggcccgg tcggcaccag ttgcgtgagc ggaaagatgg
      841 ccgcttcccg gccctgctgc agggagctca aaatggagga cgcggcgctc gggagagcgg
      901 gcgggtgagt cacccacaca aaggaaaagg gcctttccgt cctcagccgt cgcttcatgt
      961 gactccacgg agtaccgggc gccgtccagg cacctcgatt agttctgcag cttttggagt
     1021 acgtcgtctt taggttgggg ggaggggttt tatgcgatgg agtttcccca cactgagtgg
     1081 gtggagactg aagttaggcc agcttggcac ttgatgtaat tctccttgga atttgccctt
     1141 tttgagtttg gatcttggtt cattctcaag cctcagacag tggttcaaag tttttttctt
     1201 ccatttcagg tgtcgtgagg accggttata ctgcggccgc caccatggtg agcaagggcg
     1261 aggagctgtt caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc
     1321 acaagttcag cgtccgcggc gagggcgagg gcgatgccac caacggcaag ctgaccctga
     1381 agttcatctg caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccttcg
     1441 gctacggcgt ggcctgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca
     1501 agtccgccat gcccgaaggc tacgtccagg agcgcaccat ctctttcaag gacgacggta
     1561 cctacaagac ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc
     1621 tgaagggcat cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact
     1681 tcaacagcca ctacgtctat atcacggccg acaagcagaa gaactgcatc aaggctaact
     1741 tcaagatccg ccacaacgtt gaggacggca gcgtgcagct cgccgaccac taccagcaga
     1801 acacccccat cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agccatcagt
     1861 ccaagctgag caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga
     1921 ccgccgccgg gattacacat ggcatggacg agctgtacaa gtagtgacct gcaggtcgtg
     1981 tcagttatcg acaaggtcaa cgtcacctaa ataattccgc ccccccctct ccctcccccc
     2041 cccctaacgt tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt
     2101 tattttccac catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct
     2161 tcttgacgag cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga
     2221 atgtcgtgaa ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga
     2281 ccctttgcag gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac
     2341 gtgtataaga tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag
     2401 ttgtggaaag agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc
     2461 agaaggtacc ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg
     2521 tttagtcgag gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt
     2581 gaaaaacacg atgataatat ggccacaacc atgggctgca ccgtgagcgc cgaggacaag
     2641 gcggcggccg agcgcggctc tggttccggc actagtgtga gtaaaggaga ggccgttata
     2701 aaagagttca tgcggttcaa ggtacacatg gagggatcaa tgaacggaca cgagttcgaa
     2761 atcgaagggg aaggagaggg caggccctat gagggtactc agaccgccaa actcaaagtg
     2821 acaaagggag gccctttgcc tttctcttgg gacatcttgt caccacaatt tatgtatggc
     2881 tcccgagcat ttataaaaca cccagcagac atacccgatt actacaagca gtctttccca
     2941 gaaggtttta aatgggaaag ggtcatgaac tttgaggatg gtggtgctgt gacggtgacg
     3001 caggatactt ctttggagga cggaacactc atctacaagg taaagctcag gggcacgaac
     3061 ttcccgccgg acggaccagt catgcagaaa aagactatgg gatgggaggc ttccaccgaa
     3121 cggttgtatc ccgaggacgg tgttttgaag ggagatataa aaatggctct tcggctgaaa
     3181 gatggaggca gatacttggc agattttaag accacgtata aggcaaaaaa accagtgcaa
     3241 atgccgggag catataacgt tgaccgcaaa ctggacatca cctcacataa cgaggattat
     3301 acagtagtag aacagtacga aagaagcgaa gggaggcaca gcaccggggg tatggatgag
     3361 ctgtataagt gataggcatg ctagcactgg tttgaatgag gcttcagtac tttacagaat
     3421 cgttgcctgc acatcttgga aacacttgct gggattactt cgacttctta acccaacaga
     3481 aggctcgaga aggtatattg ctgttgacag tgagcgcact ctttctagga ggttgtgata
     3541 gtgaagccac agatgtatca caacctccta gaaagagtat gcctactgcc tcggacttca
     3601 aggggctaga attcgagcaa ttatcttgtt tactaaaact gaataccttg ctatctcttt
     3661 gatacatttt tacaaagctg aattaaaatg gtataaatta aatcactttt ttcaattgac
     3721 gcgtgatcta attctaccgg gtaggggagg cgcttttccc aaggcagtct ggagcatggc
     3781 cgcactcctc aggtgcaggc tgcctatcag aaggtggtgg ctggtgtggc caatgccctg
     3841 gctcacaaat accactgaga tctttttccc tctgccaaaa attatgggga catcatgaag
     3901 ccccttgagc atctgacttc tggctaataa aggaaattta ttttcattgc aatagtgtgt
     3961 tggaattttt tgtgtctctc actcggaagg acatatggga gggcaaatca tttaaaacat
     4021 cagaatgagt atttggttta gagtttggca acatatgccc atatgctggc tgccatgaac
     4081 aaaggttggc tataaagagg tcatcagtat atgaaacagc cccctgctgt ccattcctta
     4141 ttccatagaa aagccttgac ttgaggttag atttttttta tattttgttt tgtgttattt
     4201 ttttctttaa catccctaaa attttcctta catgttttac tagccagatt tttcctcctc
     4261 tcctgactac tcccagtcat agctgtccct cttctcttat ggagatccct cgacctgcag
     4321 cccaagcttg gcgtaatcat ggtcatagct gtttcctgtg tgaaattgtt atccgctcac
     4381 aattccacac aacatacgag ccggaagcat aaagtgtaaa gcctggggtg cctaatgagt
     4441 gagctaactc acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     4501 gtgccagcgg atccgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     4561 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     4621 tttatttatg cagaggccga ggccgcctcg gcctctgagc tattccagaa gtagtgagga
     4681 ggcttttttg gaggcctagg cttttgcaaa aagctaactt gtttattgca gcttataatg
     4741 gttacaaata aagcaatagc atcacaaatt tcacaaataa agcatttttt tcactgcatt
     4801 ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca tgtctggatc cgctgcatta
     4861 atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc
     4921 gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa
     4981 ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa
     5041 aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct
     5101 ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac
     5161 aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc
     5221 gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc
     5281 tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg
     5341 tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga
     5401 gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta acaggattag
     5461 cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta
     5521 cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag
     5581 agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg
     5641 caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac
     5701 ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc
     5761 aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag
     5821 tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc
     5881 agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt agataactac
     5941 gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag acccacgctc
     6001 accggctcca gatttatcag caataaacca gccagccgga agggccgagc gcagaagtgg
     6061 tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag ctagagtaag
     6121 tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc
     6181 acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa ggcgagttac
     6241 atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga tcgttgtcag
     6301 aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata attctcttac
     6361 tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca agtcattctg
     6421 agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg ataataccgc
     6481 gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact
     6541 ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg cacccaactg
     6601 atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa
     6661 tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac tcttcctttt
     6721 tcaatattat tgaagcattt atcagggtta ttgtctcatg agcggataca tatttgaatg
     6781 tatttagaaa aataaacaaa taggggttcc gcgcacattt ccccgaaaag tgccacctg
//
