LOCUS       sequence-365398-        6873 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Designed to test the efficacy of shRNA by assessing the ratio of 
            GFP-fusion protein to RFP (shRNA ratio negative control 
            (Luciferase), Fig S2).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6873)
  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 6873)
  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: shRNA-Ratio-NegCon
FEATURES             Location/Qualifiers
     source          1..6873
                     /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"
     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"
     polyA_signal    1636..1843
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     enhancer        1911..2196
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          2476..2703
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     ncRNA           3575..3589
                     /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(3895..3914)
                     /label=Bglob-pA-R
                     /note="Rabbit beta-globin polyA region, reverse primer"
     polyA_signal    3960..4015
                     /label=beta-globin poly(A) signal
                     /note="rabbit beta-globin polyadenylation signal (Gil and 
                     Proudfoot, 1987)"
     primer_bind     complement(4014..4033)
                     /label=rbglobpA-R
                     /note="Rabbit beta-globin polyA, reverse primer. Also 
                     called rb-glob-pA-term-R"
     primer_bind     complement(4376..4392)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4376..4392)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4389..4411)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4400..4416
                     /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(4424..4430,4431..4448,4449..4454))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4469..4490
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        4549..4744
                     /label=SV40 promoter
                     /note="SV40 early promoter"
     rep_origin      4595..4730
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     polyA_signal    4750..4884
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4787..4806)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4841..4860
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4952..4969)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5123..5711)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5203..5222)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5882..6673,6674..6742))
                     /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     6505..6524
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6743..6847)
                     /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 sgcgcsccgg gggggggggg gggggggggg gggggggggg gggggggggg gggggggggg
      481 ggggsrgcca awcagrgsgg sgcgcyccga aagtttcctt ttwtggcgrg gsggsggsgg
      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 ggacgagttt atctgtacct tcaaccagag tctttgatct acaaaggagc tacagatact
     1621 actttaaggc gcgccctgtg ccttctagtt gccagccatc tgttgtttgc ccctcccccg
     1681 tgccttcctt gaccctggaa ggtgccactc ccactgtcct ttcctaataa aatgaggaaa
     1741 ttgcatcgca ttgtctgagt aggtgtcatt ctattctggg gggtggggtg gggcaggaca
     1801 gcaaggggga ggattgggaa gagaatagca ggcatgatgg ggactcggta ccaaattcca
     1861 gaaaagaggc ctcccgaaag gggggccttt tttcgttttg gtccgatatc cgttacataa
     1921 cttacggtaa atggcccgcc tggctgaccg cccaacgacc cccgcccatt gacgtcaata
     1981 gtaacgccaa tagggacttt ccattgacgt caatgggtgg agtatttacg gtaaactgcc
     2041 cacttggcag tacatcaagt gtatcatatg ccaagtacgc cccctattga cgtcaatgac
     2101 ggtaaatggc ccgcctggca ttgtgcccag tacatgacct tatgggactt tcctacttgg
     2161 cagtacatct acgtattagt catcgctatt accatggtcg aggtgagccc cacgttctgc
     2221 ttcactctcc ccatctcccc ccccyccccm cccccaattt tgtatttwtt twttttttaa
     2281 ttwttttgkg crgsgrkggg ggsggggggg gggggggggc gcgcgccagg cggggcgggg
     2341 cggggcgagg ggcggggcgg ggcgaggcgg agaggtgcgg cggcagccaa tcagagcggc
     2401 gcgctccgaa agtttccttt tatggcgagg cggcggcggc ggcggcccta taaaaagcga
     2461 agcgcgcggc gggcgggagt cgctgcgcgc tgccttcgcc ccgtgccccg ctccgccgcc
     2521 gcctcgcgcc gcccgccccg gctctgactg accgcgttac tcccacaggt gagcgggcgg
     2581 gacggccctt ctcctccggg ctgtaattag ctgagcaaga ggtaagggtt taagggatgg
     2641 ttggttggtg gggtattaat gtttaattac ctggagcacc tgcctgaaat cacttttttt
     2701 caggttggac tagtatggtg agtaaaggag aggccgttat aaaagagttc atgcggttca
     2761 aggtacacat ggagggatca atgaacggac acgagttcga aatcgaaggg gaaggagagg
     2821 gcaggcccta tgagggtact cagaccgcca aactcaaagt gacaaaggga ggccctttgc
     2881 ctttctcttg ggacatcttg tcaccacaat ttatgtatgg ctcccgagca tttataaaac
     2941 acccagcaga catacccgat tactacaagc agtctttccc agaaggtttt aaatgggaaa
     3001 gggtcatgaa ctttgaggat ggtggtgctg tgacggtgac gcaggatact tctttggagg
     3061 acggaacact catctacaag gtaaagctca ggggcacgaa cttcccgccg gacggaccag
     3121 tcatgcagaa aaagactatg ggatgggagg cttccaccga acggttgtat cccgaggacg
     3181 gtgttttgaa gggagatata aaaatggctc ttcggctgaa agatggaggc agatacttgg
     3241 cagattttaa gaccacgtat aaggcaaaaa aaccagtgca aatgccggga gcatataacg
     3301 ttgaccgcaa actggacatc acctcacata acgaggatta tacagtagta gaacagtacg
     3361 aaagaagcga agggaggcac agcaccgggg gtatggatga gctgtataag tgataggcat
     3421 gctgtttgaa tgaggcttca gtactttaca gaatcgttgc ctgcacatct tggaaacact
     3481 tgctgggatt acttcgactt cttaacccaa cagaaggctc gagaaggtat attgctgttg
     3541 acagtgagcg atcatatcat tttggaatat tatagtgaag ccacagatgt ataatattcc
     3601 aaaatgatat gactgcctac tgcctcggac ttcaaggggc tagaattcga gcaattatct
     3661 tgtttactaa aactgaatac cttgctatct ctttgataca tttttacaaa gctgaattaa
     3721 aatggtataa attaaatcac ttttttcaat tgacgcgtga tctaattcta ccgggtaggg
     3781 gaggcgcttt tcccaaggca gtctggagca tggccgcact cctcaggtgc aggctgccta
     3841 tcagaaggtg gtggctggtg tggccaatgc cctggctcac aaataccact gagatctttt
     3901 tccctctgcc aaaaattatg gggacatcat gaagcccctt gagcatctga cttctggcta
     3961 ataaaggaaa tttattttca ttgcaatagt gtgttggaat tttttgtgtc tctcactcgg
     4021 aaggacatat gggagggcaa atcatttaaa acatcagaat gagtatttgg tttagagttt
     4081 ggcaacatat gcccatatgc tggctgccat gaacaaaggt tggctataaa gaggtcatca
     4141 gtatatgaaa cagccccctg ctgtccattc cttattccat agaaaagcct tgacttgagg
     4201 ttagattttt tttatatttt gttttgtgtt atttttttct ttaacatccc taaaattttc
     4261 cttacatgtt ttactagcca gatttttcct cctctcctga ctactcccag tcatagctgt
     4321 ccctcttctc ttatggagat ccctcgacct gcagcccaag cttggcgtaa tcatggtcat
     4381 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa
     4441 gcataaagtg taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc
     4501 gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca gcggatccgc atctcaatta
     4561 gtcagcaacc atagtcccgc ccctaactcc gcccatcccg cccctaactc cgcccagttc
     4621 cgcccattct ccgccccatg gctgactaat tttttttatt tatgcagagg ccgaggccgc
     4681 ctcggcctct gagctattcc agaagtagtg aggaggcttt tttggaggcc taggcttttg
     4741 caaaaagcta acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     4801 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     4861 aatgtatctt atcatgtctg gatccgctgc attaatgaat cggccaacgc gcggggagag
     4921 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg
     4981 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat
     5041 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta
     5101 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     5161 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     5221 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     5281 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     5341 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     5401 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     5461 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     5521 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     5581 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     5641 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     5701 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     5761 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     5821 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     5881 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     5941 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     6001 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     6061 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     6121 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     6181 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     6241 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     6301 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     6361 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     6421 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     6481 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     6541 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     6601 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     6661 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     6721 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     6781 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     6841 ttccgcgcac atttccccga aaagtgccac ctg
//
