LOCUS       sequence-364046-        6415 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 6415)
  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 6415)
  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.21
FEATURES             Location/Qualifiers
     source          1..6415
                     /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      851..860
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(857..859,860..862,863..1576)
                     /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(902..923)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     1510..1531
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     misc_feature    join(1607..2181,2182..2184,2185..2193)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1774..1791)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     2001..2020
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     ncRNA           3117..3131
                     /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(3437..3456)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3502..3557
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(3556..3575)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(3918..3934)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3918..3934)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3931..3953)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3942..3958
                     /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(3966..3972,3973..3990,3991..3996))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4011..4032
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4091..4286
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4137..4272
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4292..4426
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4329..4348)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4383..4402
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4494..4511)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4665..5253)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4745..4764)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5424..6215,6216..6284))
                     /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     6047..6066
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6285..6389)
                     /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 gggsgaggcg gagaggtgcg
      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 gccggagggc tatactgccc
      841 tccggcggcc gccaccatgg tgagcaaggg cgaggagctg ttcaccgggg tggtgcccat
      901 cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtccgcg gcgagggcga
      961 gggcgatgcc accaacggca agctgaccct gaagttcatc tgcaccaccg gcaagctgcc
     1021 cgtgccctgg cccaccctcg tgaccacctt cggctacggc gtggcctgct tcagccgcta
     1081 ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag gctacgtcca
     1141 ggagcgcacc atctctttca aggacgacgg tacctacaag acccgcgccg aggtgaagtt
     1201 cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca aggaggacgg
     1261 caacatcctg gggcacaagc tggagtacaa cttcaacagc cactacgtct atatcacggc
     1321 cgacaagcag aagaactgca tcaaggctaa cttcaagatc cgccacaacg ttgaggacgg
     1381 cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg gccccgtgct
     1441 gctgcccgac aaccactacc tgagccatca gtccaagctg agcaaagacc ccaacgagaa
     1501 gcgcgatcac atggtcctgc tggagttcgt gaccgccgcc gggattacac atggcatgga
     1561 cgagctgtac aagtagcctg cagggtcacc taaataattc cgcccccccc tctccctccc
     1621 ccccccctaa cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata
     1681 tgttattttc caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg
     1741 tcttcttgac gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt
     1801 tgaatgtcgt gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag
     1861 cgaccctttg caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc
     1921 cacgtgtata agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga
     1981 tagttgtgga aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg
     2041 cccagaaggt accccattgt atgggatctg atctggggcc tcggtgcaca tgctttacat
     2101 gtgtttagtc gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc
     2161 tttgaaaaac acgatgataa tatggccaca accatgggct gcaccgtgag cgccgaggac
     2221 aaggcggcgg ccgagcgcgg ctctggttcc ggcactagtg tgagtaaagg agaggccgtt
     2281 ataaaagagt tcatgcggtt caaggtacac atggagggat caatgaacgg acacgagttc
     2341 gaaatcgaag gggaaggaga gggcaggccc tatgagggta ctcagaccgc caaactcaaa
     2401 gtgacaaagg gaggcccttt gcctttctct tgggacatct tgtcaccaca atttatgtat
     2461 ggctcccgag catttataaa acacccagca gacatacccg attactacaa gcagtctttc
     2521 ccagaaggtt ttaaatggga aagggtcatg aactttgagg atggtggtgc tgtgacggtg
     2581 acgcaggata cttctttgga ggacggaaca ctcatctaca aggtaaagct caggggcacg
     2641 aacttcccgc cggacggacc agtcatgcag aaaaagacta tgggatggga ggcttccacc
     2701 gaacggttgt atcccgagga cggtgttttg aagggagata taaaaatggc tcttcggctg
     2761 aaagatggag gcagatactt ggcagatttt aagaccacgt ataaggcaaa aaaaccagtg
     2821 caaatgccgg gagcatataa cgttgaccgc aaactggaca tcacctcaca taacgaggat
     2881 tatacagtag tagaacagta cgaaagaagc gaagggaggc acagcaccgg gggtatggat
     2941 gagctgtata agtgataggc atgctgtttg aatgaggctt cagtacttta cagaatcgtt
     3001 gcctgcacat cttggaaaca cttgctggga ttacttcgac ttcttaaccc aacagaaggc
     3061 tcgagaaggt atattgctgt tgacagtgag cgcactcttt ctaggaggtt gtgatagtga
     3121 agccacagat gtatcacaac ctcctagaaa gagtatgcct actgcctcgg acttcaaggg
     3181 gctagaattc gagcaattat cttgtttact aaaactgaat accttgctat ctctttgata
     3241 catttttaca aagctgaatt aaaatggtat aaattaaatc acttttttca attgacgcgt
     3301 gatctaattc taccgggtag gggaggcgct tttcccaagg cagtctggag catggccgca
     3361 ctcctcaggt gcaggctgcc tatcagaagg tggtggctgg tgtggccaat gccctggctc
     3421 acaaatacca ctgagatctt tttccctctg ccaaaaatta tggggacatc atgaagcccc
     3481 ttgagcatct gacttctggc taataaagga aatttatttt cattgcaata gtgtgttgga
     3541 attttttgtg tctctcactc ggaaggacat atgggagggc aaatcattta aaacatcaga
     3601 atgagtattt ggtttagagt ttggcaacat atgcccatat gctggctgcc atgaacaaag
     3661 gttggctata aagaggtcat cagtatatga aacagccccc tgctgtccat tccttattcc
     3721 atagaaaagc cttgacttga ggttagattt tttttatatt ttgttttgtg ttattttttt
     3781 ctttaacatc cctaaaattt tccttacatg ttttactagc cagatttttc ctcctctcct
     3841 gactactccc agtcatagct gtccctcttc tcttatggag atccctcgac ctgcagccca
     3901 agcttggcgt aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt
     3961 ccacacaaca tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc
     4021 taactcacat taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc
     4081 cagcggatcc gcatctcaat tagtcagcaa ccatagtccc gcccctaact ccgcccatcc
     4141 cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta atttttttta
     4201 tttatgcaga ggccgaggcc gcctcggcct ctgagctatt ccagaagtag tgaggaggct
     4261 tttttggagg cctaggcttt tgcaaaaagc taacttgttt attgcagctt ataatggtta
     4321 caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac tgcattctag
     4381 ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatccgct gcattaatga
     4441 atcggccaac gcgcggggag aggcggtttg cgtattgggc gctcttccgc ttcctcgctc
     4501 actgactcgc tgcgctcggt cgttcggctg cggcgagcgg tatcagctca ctcaaaggcg
     4561 gtaatacggt tatccacaga atcaggggat aacgcaggaa agaacatgtg agcaaaaggc
     4621 cagcaaaagg ccaggaaccg taaaaaggcc gcgttgctgg cgtttttcca taggctccgc
     4681 ccccctgacg agcatcacaa aaatcgacgc tcaagtcaga ggtggcgaaa cccgacagga
     4741 ctataaagat accaggcgtt tccccctgga agctccctcg tgcgctctcc tgttccgacc
     4801 ctgccgctta ccggatacct gtccgccttt ctcccttcgg gaagcgtggc gctttctcat
     4861 agctcacgct gtaggtatct cagttcggtg taggtcgttc gctccaagct gggctgtgtg
     4921 cacgaacccc ccgttcagcc cgaccgctgc gccttatccg gtaactatcg tcttgagtcc
     4981 aacccggtaa gacacgactt atcgccactg gcagcagcca ctggtaacag gattagcaga
     5041 gcgaggtatg taggcggtgc tacagagttc ttgaagtggt ggcctaacta cggctacact
     5101 agaagaacag tatttggtat ctgcgctctg ctgaagccag ttaccttcgg aaaaagagtt
     5161 ggtagctctt gatccggcaa acaaaccacc gctggtagcg gtggtttttt tgtttgcaag
     5221 cagcagatta cgcgcagaaa aaaaggatct caagaagatc ctttgatctt ttctacgggg
     5281 tctgacgctc agtggaacga aaactcacgt taagggattt tggtcatgag attatcaaaa
     5341 aggatcttca cctagatcct tttaaattaa aaatgaagtt ttaaatcaat ctaaagtata
     5401 tatgagtaaa cttggtctga cagttaccaa tgcttaatca gtgaggcacc tatctcagcg
     5461 atctgtctat ttcgttcatc catagttgcc tgactccccg tcgtgtagat aactacgata
     5521 cgggagggct taccatctgg ccccagtgct gcaatgatac cgcgagaccc acgctcaccg
     5581 gctccagatt tatcagcaat aaaccagcca gccggaaggg ccgagcgcag aagtggtcct
     5641 gcaactttat ccgcctccat ccagtctatt aattgttgcc gggaagctag agtaagtagt
     5701 tcgccagtta atagtttgcg caacgttgtt gccattgcta caggcatcgt ggtgtcacgc
     5761 tcgtcgtttg gtatggcttc attcagctcc ggttcccaac gatcaaggcg agttacatga
     5821 tcccccatgt tgtgcaaaaa agcggttagc tccttcggtc ctccgatcgt tgtcagaagt
     5881 aagttggccg cagtgttatc actcatggtt atggcagcac tgcataattc tcttactgtc
     5941 atgccatccg taagatgctt ttctgtgact ggtgagtact caaccaagtc attctgagaa
     6001 tagtgtatgc ggcgaccgag ttgctcttgc ccggcgtcaa tacgggataa taccgcgcca
     6061 catagcagaa ctttaaaagt gctcatcatt ggaaaacgtt cttcggggcg aaaactctca
     6121 aggatcttac cgctgttgag atccagttcg atgtaaccca ctcgtgcacc caactgatct
     6181 tcagcatctt ttactttcac cagcgtttct gggtgagcaa aaacaggaag gcaaaatgcc
     6241 gcaaaaaagg gaataagggc gacacggaaa tgttgaatac tcatactctt cctttttcaa
     6301 tattattgaa gcatttatca gggttattgt ctcatgagcg gatacatatt tgaatgtatt
     6361 tagaaaaata aacaaatagg ggttccgcgc acatttcccc gaaaagtgcc acctg
//
