LOCUS       sequence-365373-        6822 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 6822)
  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 6822)
  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.5
FEATURES             Location/Qualifiers
     source          1..6822
                     /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      1258..1267
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1264..1266,1267..1269,1270..1983)
                     /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(1309..1330)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1917..1938
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(2014..2588,2589..2591,2592..2600)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(2181..2198)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2408..2427
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     ncRNA           3524..3538
                     /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(3844..3863)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3909..3964
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3963..3982)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4325..4341)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4325..4341)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4338..4360)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4349..4365
                     /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(4373..4379,4380..4397,4398..4403))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4418..4439
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4498..4693
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4544..4679
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4699..4833
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4736..4755)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4790..4809
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4901..4918)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5072..5660)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5152..5171)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5831..6622,6623..6691))
                     /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     6454..6473
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6692..6796)
                     /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 cgcgcgacgt atactcgtcg cgcggccgcc
     1261 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     1321 gacggcgacg taaacggcca caagttcagc gtccgcggcg agggcgaggg cgatgccacc
     1381 aacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     1441 accctcgtga ccaccttcgg ctacggcgtg gcctgcttca gccgctaccc cgaccacatg
     1501 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     1561 tctttcaagg acgacggtac ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     1621 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     1681 cacaagctgg agtacaactt caacagccac tacgtctata tcacggccga caagcagaag
     1741 aactgcatca aggctaactt caagatccgc cacaacgttg aggacggcag cgtgcagctc
     1801 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     1861 cactacctga gccatcagtc caagctgagc aaagacccca acgagaagcg cgatcacatg
     1921 gtcctgctgg agttcgtgac cgccgccggg attacacatg gcatggacga gctgtacaag
     1981 tagcctgcag ggtcacctaa ataattccgc ccccccctct ccctcccccc cccctaacgt
     2041 tactggccga agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac
     2101 catattgccg tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag
     2161 cattcctagg ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa
     2221 ggaagcagtt cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag
     2281 gcagcggaac cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga
     2341 tacacctgca aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag
     2401 agtcaaatgg ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc
     2461 ccattgtatg ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag
     2521 gttaaaaaaa cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg
     2581 atgataatat ggccacaacc atgggctgca ccgtgagcgc cgaggacaag gcggcggccg
     2641 agcgcggctc tggttccggc actagtgtga gtaaaggaga ggccgttata aaagagttca
     2701 tgcggttcaa ggtacacatg gagggatcaa tgaacggaca cgagttcgaa atcgaagggg
     2761 aaggagaggg caggccctat gagggtactc agaccgccaa actcaaagtg acaaagggag
     2821 gccctttgcc tttctcttgg gacatcttgt caccacaatt tatgtatggc tcccgagcat
     2881 ttataaaaca cccagcagac atacccgatt actacaagca gtctttccca gaaggtttta
     2941 aatgggaaag ggtcatgaac tttgaggatg gtggtgctgt gacggtgacg caggatactt
     3001 ctttggagga cggaacactc atctacaagg taaagctcag gggcacgaac ttcccgccgg
     3061 acggaccagt catgcagaaa aagactatgg gatgggaggc ttccaccgaa cggttgtatc
     3121 ccgaggacgg tgttttgaag ggagatataa aaatggctct tcggctgaaa gatggaggca
     3181 gatacttggc agattttaag accacgtata aggcaaaaaa accagtgcaa atgccgggag
     3241 catataacgt tgaccgcaaa ctggacatca cctcacataa cgaggattat acagtagtag
     3301 aacagtacga aagaagcgaa gggaggcaca gcaccggggg tatggatgag ctgtataagt
     3361 gataggcatg ctgtttgaat gaggcttcag tactttacag aatcgttgcc tgcacatctt
     3421 ggaaacactt gctgggatta cttcgacttc ttaacccaac agaaggctcg agaaggtata
     3481 ttgctgttga cagtgagcgc actctttcta ggaggttgtg atagtgaagc cacagatgta
     3541 tcacaacctc ctagaaagag tatgcctact gcctcggact tcaaggggct agaattcgag
     3601 caattatctt gtttactaaa actgaatacc ttgctatctc tttgatacat ttttacaaag
     3661 ctgaattaaa atggtataaa ttaaatcact tttttcaatt gacgcgtgat ctaattctac
     3721 cgggtagggg aggcgctttt cccaaggcag tctggagcat ggccgcactc ctcaggtgca
     3781 ggctgcctat cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg
     3841 agatcttttt ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac
     3901 ttctggctaa taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct
     3961 ctcactcgga aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt
     4021 ttagagtttg gcaacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag
     4081 aggtcatcag tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt
     4141 gacttgaggt tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct
     4201 aaaattttcc ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt
     4261 catagctgtc cctcttctct tatggagatc cctcgacctg cagcccaagc ttggcgtaat
     4321 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     4381 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     4441 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatccgca
     4501 tctcaattag tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc
     4561 gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc
     4621 cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct
     4681 aggcttttgc aaaaagctaa cttgtttatt gcagcttata atggttacaa ataaagcaat
     4741 agcatcacaa atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc
     4801 aaactcatca atgtatctta tcatgtctgg atccgctgca ttaatgaatc ggccaacgcg
     4861 cggggagagg cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc
     4921 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat
     4981 ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca
     5041 ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc
     5101 atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc
     5161 aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
     5221 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta
     5281 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg
     5341 ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac
     5401 acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag
     5461 gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat
     5521 ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
     5581 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc
     5641 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt
     5701 ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct
     5761 agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt
     5821 ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc
     5881 gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
     5941 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat
     6001 cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg
     6061 cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata
     6121 gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta
     6181 tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt
     6241 gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
     6301 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa
     6361 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc
     6421 gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt
     6481 taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc
     6541 tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta
     6601 ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
     6661 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca
     6721 tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac
     6781 aaataggggt tccgcgcaca tttccccgaa aagtgccacc tg
//
