LOCUS       sequence-365457-        6392 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 CBh promoter).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6392)
  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 6392)
  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-CBh
FEATURES             Location/Qualifiers
     source          1..6392
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        12..297
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     promoter        299..576
                     /label=chicken beta-actin promoter
     intron          577..804
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      828..837
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(834..836,837..839,840..1553)
                     /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(879..900)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1487..1508
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(1584..2158,2159..2161,2162..2170)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1751..1768)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1978..1997
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     ncRNA           3094..3108
                     /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(3414..3433)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3479..3534
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3533..3552)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3895..3911)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3895..3911)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3908..3930)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3919..3935
                     /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(3943..3949,3950..3967,3968..3973))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3988..4009
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4068..4263
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4114..4249
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4269..4403
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4306..4325)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4360..4379
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4471..4488)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4642..5230)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4722..4741)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5401..6192,6193..6261))
                     /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     6024..6043
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6262..6366)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 gtcgacgtac ccgttacata acttacggta aatggcccgc ctggctgacc gcccaacgac
       61 ccccgcccat tgacgtcaat agtaacgcca atagggactt tccattgacg tcaatgggtg
      121 gagtatttac ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg
      181 ccccctattg acgtcaatga cggtaaatgg cccgcctggc attgtgccca gtacatgacc
      241 ttatgggact ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtc
      301 gaggtgagcc ccacgttctg cttcactctc cccatctccc ccccctcccc acccccaatt
      361 ttgtatttat ttatttttta attattttgt gcagcgatgg gggcgggggg gggggggggg
      421 cgcgcgccrg gsggggsggg gsggggsgrg gggsggggsg gggsgrggcg gagaggkgcg
      481 gcggcagcca atcagagcgg cgcgctccga aagtttcctt ttatggcgag gcggcggcgg
      541 cggcggccct ataaaaagcg aagcgcgcgg cgggcgggag tcgctgcgcg ctgccttcgc
      601 cccgtgcccc gctccgccgc cgcctcgcgc cgcccgcccc ggctctgact gaccgcgtta
      661 ctcccacagg tgagcgggcg ggacggccct tctcctccgg gctgtaatta gctgagcaag
      721 aggtaagggt ttaagggatg gttggttggt ggggtattaa tgtttaatta cctggagcac
      781 ctgcctgaaa tcactttttt tcaggttgga ccggttatac tgcggccgcc accatggtga
      841 gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg
      901 taaacggcca caagttcagc gtccgcggcg agggcgaggg cgatgccacc aacggcaagc
      961 tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga
     1021 ccaccttcgg ctacggcgtg gcctgcttca gccgctaccc cgaccacatg aagcagcacg
     1081 acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc tctttcaagg
     1141 acgacggtac ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc
     1201 gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg
     1261 agtacaactt caacagccac tacgtctata tcacggccga caagcagaag aactgcatca
     1321 aggctaactt caagatccgc cacaacgttg aggacggcag cgtgcagctc gccgaccact
     1381 accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga
     1441 gccatcagtc caagctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg
     1501 agttcgtgac cgccgccggg attacacatg gcatggacga gctgtacaag tagcctgcag
     1561 ggtcacctaa ataattccgc ccccccctct ccctcccccc cccctaacgt tactggccga
     1621 agccgcttgg aataaggccg gtgtgcgttt gtctatatgt tattttccac catattgccg
     1681 tcttttggca atgtgagggc ccggaaacct ggccctgtct tcttgacgag cattcctagg
     1741 ggtctttccc ctctcgccaa aggaatgcaa ggtctgttga atgtcgtgaa ggaagcagtt
     1801 cctctggaag cttcttgaag acaaacaacg tctgtagcga ccctttgcag gcagcggaac
     1861 cccccacctg gcgacaggtg cctctgcggc caaaagccac gtgtataaga tacacctgca
     1921 aaggcggcac aaccccagtg ccacgttgtg agttggatag ttgtggaaag agtcaaatgg
     1981 ctctcctcaa gcgtattcaa caaggggctg aaggatgccc agaaggtacc ccattgtatg
     2041 ggatctgatc tggggcctcg gtgcacatgc tttacatgtg tttagtcgag gttaaaaaaa
     2101 cgtctaggcc ccccgaacca cggggacgtg gttttccttt gaaaaacacg atgataatat
     2161 ggccacaacc atgggctgca ccgtgagcgc cgaggacaag gcggcggccg agcgcggctc
     2221 tggttccggc actagtgtga gtaaaggaga ggccgttata aaagagttca tgcggttcaa
     2281 ggtacacatg gagggatcaa tgaacggaca cgagttcgaa atcgaagggg aaggagaggg
     2341 caggccctat gagggtactc agaccgccaa actcaaagtg acaaagggag gccctttgcc
     2401 tttctcttgg gacatcttgt caccacaatt tatgtatggc tcccgagcat ttataaaaca
     2461 cccagcagac atacccgatt actacaagca gtctttccca gaaggtttta aatgggaaag
     2521 ggtcatgaac tttgaggatg gtggtgctgt gacggtgacg caggatactt ctttggagga
     2581 cggaacactc atctacaagg taaagctcag gggcacgaac ttcccgccgg acggaccagt
     2641 catgcagaaa aagactatgg gatgggaggc ttccaccgaa cggttgtatc ccgaggacgg
     2701 tgttttgaag ggagatataa aaatggctct tcggctgaaa gatggaggca gatacttggc
     2761 agattttaag accacgtata aggcaaaaaa accagtgcaa atgccgggag catataacgt
     2821 tgaccgcaaa ctggacatca cctcacataa cgaggattat acagtagtag aacagtacga
     2881 aagaagcgaa gggaggcaca gcaccggggg tatggatgag ctgtataagt gataggcatg
     2941 ctgtttgaat gaggcttcag tactttacag aatcgttgcc tgcacatctt ggaaacactt
     3001 gctgggatta cttcgacttc ttaacccaac agaaggctcg agaaggtata ttgctgttga
     3061 cagtgagcgc actctttcta ggaggttgtg atagtgaagc cacagatgta tcacaacctc
     3121 ctagaaagag tatgcctact gcctcggact tcaaggggct agaattcgag caattatctt
     3181 gtttactaaa actgaatacc ttgctatctc tttgatacat ttttacaaag ctgaattaaa
     3241 atggtataaa ttaaatcact tttttcaatt gacgcgtgat ctaattctac cgggtagggg
     3301 aggcgctttt cccaaggcag tctggagcat ggccgcactc ctcaggtgca ggctgcctat
     3361 cagaaggtgg tggctggtgt ggccaatgcc ctggctcaca aataccactg agatcttttt
     3421 ccctctgcca aaaattatgg ggacatcatg aagccccttg agcatctgac ttctggctaa
     3481 taaaggaaat ttattttcat tgcaatagtg tgttggaatt ttttgtgtct ctcactcgga
     3541 aggacatatg ggagggcaaa tcatttaaaa catcagaatg agtatttggt ttagagtttg
     3601 gcaacatatg cccatatgct ggctgccatg aacaaaggtt ggctataaag aggtcatcag
     3661 tatatgaaac agccccctgc tgtccattcc ttattccata gaaaagcctt gacttgaggt
     3721 tagatttttt ttatattttg ttttgtgtta tttttttctt taacatccct aaaattttcc
     3781 ttacatgttt tactagccag atttttcctc ctctcctgac tactcccagt catagctgtc
     3841 cctcttctct tatggagatc cctcgacctg cagcccaagc ttggcgtaat catggtcata
     3901 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     3961 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     4021 ctcactgccc gctttccagt cgggaaacct gtcgtgccag cggatccgca tctcaattag
     4081 tcagcaacca tagtcccgcc cctaactccg cccatcccgc ccctaactcc gcccagttcc
     4141 gcccattctc cgccccatgg ctgactaatt ttttttattt atgcagaggc cgaggccgcc
     4201 tcggcctctg agctattcca gaagtagtga ggaggctttt ttggaggcct aggcttttgc
     4261 aaaaagctaa cttgtttatt gcagcttata atggttacaa ataaagcaat agcatcacaa
     4321 atttcacaaa taaagcattt ttttcactgc attctagttg tggtttgtcc aaactcatca
     4381 atgtatctta tcatgtctgg atccgctgca ttaatgaatc ggccaacgcg cggggagagg
     4441 cggtttgcgt attgggcgct cttccgcttc ctcgctcact gactcgctgc gctcggtcgt
     4501 tcggctgcgg cgagcggtat cagctcactc aaaggcggta atacggttat ccacagaatc
     4561 aggggataac gcaggaaaga acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa
     4621 aaaggccgcg ttgctggcgt ttttccatag gctccgcccc cctgacgagc atcacaaaaa
     4681 tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc aggcgtttcc
     4741 ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg gatacctgtc
     4801 cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc tcacgctgta ggtatctcag
     4861 ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga
     4921 ccgctgcgcc ttatccggta actatcgtct tgagtccaac ccggtaagac acgacttatc
     4981 gccactggca gcagccactg gtaacaggat tagcagagcg aggtatgtag gcggtgctac
     5041 agagttcttg aagtggtggc ctaactacgg ctacactaga agaacagtat ttggtatctg
     5101 cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat ccggcaaaca
     5161 aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa
     5221 aggatctcaa gaagatcctt tgatcttttc tacggggtct gacgctcagt ggaacgaaaa
     5281 ctcacgttaa gggattttgg tcatgagatt atcaaaaagg atcttcacct agatcctttt
     5341 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     5401 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     5461 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     5521 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     5581 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     5641 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     5701 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     5761 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     5821 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     5881 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     5941 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     6001 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     6061 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     6121 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     6181 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     6241 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     6301 ttattgtctc atgagcggat acatatttga atgtatttag aaaaataaac aaataggggt
     6361 tccgcgcaca tttccccgaa aagtgccacc tg
//
