LOCUS       sequence-365377-        6824 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 (attenuated EF1a promoter, Fig 3).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6824)
  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 6824)
  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.12
FEATURES             Location/Qualifiers
     source          1..6824
                     /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      1260..1269
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1266..1268,1269..1271,1272..1985)
                     /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(1311..1332)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1919..1940
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(2016..2590,2591..2593,2594..2602)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2183..2200)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2410..2429
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     ncRNA           3526..3540
                     /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(3846..3865)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3911..3966
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3965..3984)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4327..4343)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4327..4343)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4340..4362)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4351..4367
                     /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(4375..4381,4382..4399,4400..4405))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4420..4441
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4500..4695
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4546..4681
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4701..4835
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4738..4757)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4792..4811
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4903..4920)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5074..5662)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5154..5173)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5833..6624,6625..6693))
                     /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     6456..6475
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6694..6798)
                     /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 accggtcggc cgcggtgcgg tatactccgc accgcggccg
     1261 ccaccatggt gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc
     1321 tggacggcga cgtaaacggc cacaagttca gcgtccgcgg cgagggcgag ggcgatgcca
     1381 ccaacggcaa gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc
     1441 ccaccctcgt gaccaccttc ggctacggcg tggcctgctt cagccgctac cccgaccaca
     1501 tgaagcagca cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca
     1561 tctctttcaa ggacgacggt acctacaaga cccgcgccga ggtgaagttc gagggcgaca
     1621 ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg
     1681 ggcacaagct ggagtacaac ttcaacagcc actacgtcta tatcacggcc gacaagcaga
     1741 agaactgcat caaggctaac ttcaagatcc gccacaacgt tgaggacggc agcgtgcagc
     1801 tcgccgacca ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca
     1861 accactacct gagccatcag tccaagctga gcaaagaccc caacgagaag cgcgatcaca
     1921 tggtcctgct ggagttcgtg accgccgccg ggattacaca tggcatggac gagctgtaca
     1981 agtagcctgc agggtcacct aaataattcc gcccccccct ctccctcccc cccccctaac
     2041 gttactggcc gaagccgctt ggaataaggc cggtgtgcgt ttgtctatat gttattttcc
     2101 accatattgc cgtcttttgg caatgtgagg gcccggaaac ctggccctgt cttcttgacg
     2161 agcattccta ggggtctttc ccctctcgcc aaaggaatgc aaggtctgtt gaatgtcgtg
     2221 aaggaagcag ttcctctgga agcttcttga agacaaacaa cgtctgtagc gaccctttgc
     2281 aggcagcgga accccccacc tggcgacagg tgcctctgcg gccaaaagcc acgtgtataa
     2341 gatacacctg caaaggcggc acaaccccag tgccacgttg tgagttggat agttgtggaa
     2401 agagtcaaat ggctctcctc aagcgtattc aacaaggggc tgaaggatgc ccagaaggta
     2461 ccccattgta tgggatctga tctggggcct cggtgcacat gctttacatg tgtttagtcg
     2521 aggttaaaaa aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca
     2581 cgatgataat atggccacaa ccatgggctg caccgtgagc gccgaggaca aggcggcggc
     2641 cgagcgcggc tctggttccg gcactagtgt gagtaaagga gaggccgtta taaaagagtt
     2701 catgcggttc aaggtacaca tggagggatc aatgaacgga cacgagttcg aaatcgaagg
     2761 ggaaggagag ggcaggccct atgagggtac tcagaccgcc aaactcaaag tgacaaaggg
     2821 aggccctttg cctttctctt gggacatctt gtcaccacaa tttatgtatg gctcccgagc
     2881 atttataaaa cacccagcag acatacccga ttactacaag cagtctttcc cagaaggttt
     2941 taaatgggaa agggtcatga actttgagga tggtggtgct gtgacggtga cgcaggatac
     3001 ttctttggag gacggaacac tcatctacaa ggtaaagctc aggggcacga acttcccgcc
     3061 ggacggacca gtcatgcaga aaaagactat gggatgggag gcttccaccg aacggttgta
     3121 tcccgaggac ggtgttttga agggagatat aaaaatggct cttcggctga aagatggagg
     3181 cagatacttg gcagatttta agaccacgta taaggcaaaa aaaccagtgc aaatgccggg
     3241 agcatataac gttgaccgca aactggacat cacctcacat aacgaggatt atacagtagt
     3301 agaacagtac gaaagaagcg aagggaggca cagcaccggg ggtatggatg agctgtataa
     3361 gtgataggca tgctgtttga atgaggcttc agtactttac agaatcgttg cctgcacatc
     3421 ttggaaacac ttgctgggat tacttcgact tcttaaccca acagaaggct cgagaaggta
     3481 tattgctgtt gacagtgagc gcactctttc taggaggttg tgatagtgaa gccacagatg
     3541 tatcacaacc tcctagaaag agtatgccta ctgcctcgga cttcaagggg ctagaattcg
     3601 agcaattatc ttgtttacta aaactgaata ccttgctatc tctttgatac atttttacaa
     3661 agctgaatta aaatggtata aattaaatca cttttttcaa ttgacgcgtg atctaattct
     3721 accgggtagg ggaggcgctt ttcccaaggc agtctggagc atggccgcac tcctcaggtg
     3781 caggctgcct atcagaaggt ggtggctggt gtggccaatg ccctggctca caaataccac
     3841 tgagatcttt ttccctctgc caaaaattat ggggacatca tgaagcccct tgagcatctg
     3901 acttctggct aataaaggaa atttattttc attgcaatag tgtgttggaa ttttttgtgt
     3961 ctctcactcg gaaggacata tgggagggca aatcatttaa aacatcagaa tgagtatttg
     4021 gtttagagtt tggcaacata tgcccatatg ctggctgcca tgaacaaagg ttggctataa
     4081 agaggtcatc agtatatgaa acagccccct gctgtccatt ccttattcca tagaaaagcc
     4141 ttgacttgag gttagatttt ttttatattt tgttttgtgt tatttttttc tttaacatcc
     4201 ctaaaatttt ccttacatgt tttactagcc agatttttcc tcctctcctg actactccca
     4261 gtcatagctg tccctcttct cttatggaga tccctcgacc tgcagcccaa gcttggcgta
     4321 atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat
     4381 acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct aactcacatt
     4441 aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc agcggatccg
     4501 catctcaatt agtcagcaac catagtcccg cccctaactc cgcccatccc gcccctaact
     4561 ccgcccagtt ccgcccattc tccgccccat ggctgactaa ttttttttat ttatgcagag
     4621 gccgaggccg cctcggcctc tgagctattc cagaagtagt gaggaggctt ttttggaggc
     4681 ctaggctttt gcaaaaagct aacttgttta ttgcagctta taatggttac aaataaagca
     4741 atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt tgtggtttgt
     4801 ccaaactcat caatgtatct tatcatgtct ggatccgctg cattaatgaa tcggccaacg
     4861 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     4921 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     4981 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc
     5041 caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga
     5101 gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata
     5161 ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac
     5221 cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg
     5281 taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc
     5341 cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag
     5401 acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt
     5461 aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaagaacagt
     5521 atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg
     5581 atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac
     5641 gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca
     5701 gtggaacgaa aactcacgtt aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac
     5761 ctagatcctt ttaaattaaa aatgaagttt taaatcaatc taaagtatat atgagtaaac
     5821 ttggtctgac agttaccaat gcttaatcag tgaggcacct atctcagcga tctgtctatt
     5881 tcgttcatcc atagttgcct gactccccgt cgtgtagata actacgatac gggagggctt
     5941 accatctggc cccagtgctg caatgatacc gcgagaccca cgctcaccgg ctccagattt
     6001 atcagcaata aaccagccag ccggaagggc cgagcgcaga agtggtcctg caactttatc
     6061 cgcctccatc cagtctatta attgttgccg ggaagctaga gtaagtagtt cgccagttaa
     6121 tagtttgcgc aacgttgttg ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg
     6181 tatggcttca ttcagctccg gttcccaacg atcaaggcga gttacatgat cccccatgtt
     6241 gtgcaaaaaa gcggttagct ccttcggtcc tccgatcgtt gtcagaagta agttggccgc
     6301 agtgttatca ctcatggtta tggcagcact gcataattct cttactgtca tgccatccgt
     6361 aagatgcttt tctgtgactg gtgagtactc aaccaagtca ttctgagaat agtgtatgcg
     6421 gcgaccgagt tgctcttgcc cggcgtcaat acgggataat accgcgccac atagcagaac
     6481 tttaaaagtg ctcatcattg gaaaacgttc ttcggggcga aaactctcaa ggatcttacc
     6541 gctgttgaga tccagttcga tgtaacccac tcgtgcaccc aactgatctt cagcatcttt
     6601 tactttcacc agcgtttctg ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg
     6661 aataagggcg acacggaaat gttgaatact catactcttc ctttttcaat attattgaag
     6721 catttatcag ggttattgtc tcatgagcgg atacatattt gaatgtattt agaaaaataa
     6781 acaaataggg gttccgcgca catttccccg aaaagtgcca cctg
//
