LOCUS       sequence-370310-        6423 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 CBh promoter, Fig 3).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6423)
  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 6423)
  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.22
FEATURES             Location/Qualifiers
     source          1..6423
                     /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      859..868
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(865..867,868..870,871..1584)
                     /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(910..931)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1518..1539
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(1615..2189,2190..2192,2193..2201)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1782..1799)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2009..2028
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     ncRNA           3125..3139
                     /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(3445..3464)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3510..3565
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3564..3583)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3926..3942)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3926..3942)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3939..3961)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3950..3966
                     /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(3974..3980,3981..3998,3999..4004))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4019..4040
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4099..4294
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4145..4280
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4300..4434
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4337..4356)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4391..4410
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4502..4519)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4673..5261)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4753..4772)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5432..6223,6224..6292))
                     /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     6055..6074
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6293..6397)
                     /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 ccggtcggcc gcccagcccc tatactgggg
      841 ctgggcggcc gcccagccgc caccatggtg agcaagggcg aggagctgtt caccggggtg
      901 gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag cgtccgcggc
      961 gagggcgagg gcgatgccac caacggcaag ctgaccctga agttcatctg caccaccggc
     1021 aagctgcccg tgccctggcc caccctcgtg accaccttcg gctacggcgt ggcctgcttc
     1081 agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat gcccgaaggc
     1141 tacgtccagg agcgcaccat ctctttcaag gacgacggta cctacaagac ccgcgccgag
     1201 gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat cgacttcaag
     1261 gaggacggca acatcctggg gcacaagctg gagtacaact tcaacagcca ctacgtctat
     1321 atcacggccg acaagcagaa gaactgcatc aaggctaact tcaagatccg ccacaacgtt
     1381 gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat cggcgacggc
     1441 cccgtgctgc tgcccgacaa ccactacctg agccatcagt ccaagctgag caaagacccc
     1501 aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg gattacacat
     1561 ggcatggacg agctgtacaa gtagcctgca gggtcaccta aataattccg cccccccctc
     1621 tccctccccc ccccctaacg ttactggccg aagccgcttg gaataaggcc ggtgtgcgtt
     1681 tgtctatatg ttattttcca ccatattgcc gtcttttggc aatgtgaggg cccggaaacc
     1741 tggccctgtc ttcttgacga gcattcctag gggtctttcc cctctcgcca aaggaatgca
     1801 aggtctgttg aatgtcgtga aggaagcagt tcctctggaa gcttcttgaa gacaaacaac
     1861 gtctgtagcg accctttgca ggcagcggaa ccccccacct ggcgacaggt gcctctgcgg
     1921 ccaaaagcca cgtgtataag atacacctgc aaaggcggca caaccccagt gccacgttgt
     1981 gagttggata gttgtggaaa gagtcaaatg gctctcctca agcgtattca acaaggggct
     2041 gaaggatgcc cagaaggtac cccattgtat gggatctgat ctggggcctc ggtgcacatg
     2101 ctttacatgt gtttagtcga ggttaaaaaa acgtctaggc cccccgaacc acggggacgt
     2161 ggttttcctt tgaaaaacac gatgataata tggccacaac catgggctgc accgtgagcg
     2221 ccgaggacaa ggcggcggcc gagcgcggct ctggttccgg cactagtgtg agtaaaggag
     2281 aggccgttat aaaagagttc atgcggttca aggtacacat ggagggatca atgaacggac
     2341 acgagttcga aatcgaaggg gaaggagagg gcaggcccta tgagggtact cagaccgcca
     2401 aactcaaagt gacaaaggga ggccctttgc ctttctcttg ggacatcttg tcaccacaat
     2461 ttatgtatgg ctcccgagca tttataaaac acccagcaga catacccgat tactacaagc
     2521 agtctttccc agaaggtttt aaatgggaaa gggtcatgaa ctttgaggat ggtggtgctg
     2581 tgacggtgac gcaggatact tctttggagg acggaacact catctacaag gtaaagctca
     2641 ggggcacgaa cttcccgccg gacggaccag tcatgcagaa aaagactatg ggatgggagg
     2701 cttccaccga acggttgtat cccgaggacg gtgttttgaa gggagatata aaaatggctc
     2761 ttcggctgaa agatggaggc agatacttgg cagattttaa gaccacgtat aaggcaaaaa
     2821 aaccagtgca aatgccggga gcatataacg ttgaccgcaa actggacatc acctcacata
     2881 acgaggatta tacagtagta gaacagtacg aaagaagcga agggaggcac agcaccgggg
     2941 gtatggatga gctgtataag tgataggcat gctgtttgaa tgaggcttca gtactttaca
     3001 gaatcgttgc ctgcacatct tggaaacact tgctgggatt acttcgactt cttaacccaa
     3061 cagaaggctc gagaaggtat attgctgttg acagtgagcg cactctttct aggaggttgt
     3121 gatagtgaag ccacagatgt atcacaacct cctagaaaga gtatgcctac tgcctcggac
     3181 ttcaaggggc tagaattcga gcaattatct tgtttactaa aactgaatac cttgctatct
     3241 ctttgataca tttttacaaa gctgaattaa aatggtataa attaaatcac ttttttcaat
     3301 tgacgcgtga tctaattcta ccgggtaggg gaggcgcttt tcccaaggca gtctggagca
     3361 tggccgcact cctcaggtgc aggctgccta tcagaaggtg gtggctggtg tggccaatgc
     3421 cctggctcac aaataccact gagatctttt tccctctgcc aaaaattatg gggacatcat
     3481 gaagcccctt gagcatctga cttctggcta ataaaggaaa tttattttca ttgcaatagt
     3541 gtgttggaat tttttgtgtc tctcactcgg aaggacatat gggagggcaa atcatttaaa
     3601 acatcagaat gagtatttgg tttagagttt ggcaacatat gcccatatgc tggctgccat
     3661 gaacaaaggt tggctataaa gaggtcatca gtatatgaaa cagccccctg ctgtccattc
     3721 cttattccat agaaaagcct tgacttgagg ttagattttt tttatatttt gttttgtgtt
     3781 atttttttct ttaacatccc taaaattttc cttacatgtt ttactagcca gatttttcct
     3841 cctctcctga ctactcccag tcatagctgt ccctcttctc ttatggagat ccctcgacct
     3901 gcagcccaag cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc
     3961 tcacaattcc acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat
     4021 gagtgagcta actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc
     4081 tgtcgtgcca gcggatccgc atctcaatta gtcagcaacc atagtcccgc ccctaactcc
     4141 gcccatcccg cccctaactc cgcccagttc cgcccattct ccgccccatg gctgactaat
     4201 tttttttatt tatgcagagg ccgaggccgc ctcggcctct gagctattcc agaagtagtg
     4261 aggaggcttt tttggaggcc taggcttttg caaaaagcta acttgtttat tgcagcttat
     4321 aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt tttttcactg
     4381 cattctagtt gtggtttgtc caaactcatc aatgtatctt atcatgtctg gatccgctgc
     4441 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc tcttccgctt
     4501 cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta tcagctcact
     4561 caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag aacatgtgag
     4621 caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg tttttccata
     4681 ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg tggcgaaacc
     4741 cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg cgctctcctg
     4801 ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga agcgtggcgc
     4861 tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc tccaagctgg
     4921 gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt aactatcgtc
     4981 ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact ggtaacagga
     5041 ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg cctaactacg
     5101 gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt accttcggaa
     5161 aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt ggtttttttg
     5221 tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct ttgatctttt
     5281 ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg gtcatgagat
     5341 tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt aaatcaatct
     5401 aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt gaggcaccta
     5461 tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc gtgtagataa
     5521 ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg cgagacccac
     5581 gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc gagcgcagaa
     5641 gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg gaagctagag
     5701 taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca ggcatcgtgg
     5761 tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga tcaaggcgag
     5821 ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct ccgatcgttg
     5881 tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg cataattctc
     5941 ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca accaagtcat
     6001 tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata cgggataata
     6061 ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct tcggggcgaa
     6121 aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact cgtgcaccca
     6181 actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa acaggaaggc
     6241 aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc atactcttcc
     6301 tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga tacatatttg
     6361 aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga aaagtgccac
     6421 ctg
//
