LOCUS       sequence-224101-        5354 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For one-step cloning (with blue/ white screening) of a desired 5' 
            UTR to test its impact on translation of a firefly luciferase 
            reporter.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5354)
  AUTHORS   Jewer M, Lee L, Leibovitch M, Zhang G, Liu J, Findlay SD, Vincent 
            KM, Tandoc K, Dieters-Castator D, Quail DF, Dutta I, Coatham M, Xu 
            Z, Puri A, Guan BJ, Hatzoglou M, Brumwell A, Uniacke J, Patsis C, 
            Koromilas A, Schueler J, Siegers GM, Topisirovic I, Postovit LM
  TITLE     Translational control of breast cancer plasticity.
  JOURNAL   Nat Commun. 2020 May 19;11(1):2498. doi: 10.1038/s41467-020-16352-z.
  PUBMED    32427827
REFERENCE   2  (bases 1 to 5354)
  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: "Nat 
            Commun."; date: "2020-05-19"; volume: "11(1):2498. doi"; pages: " 
            10.1038/s41467-020-16352-z"
COMMENT     Sequence Label: pGL3-TK-5UTR-BsmBI-Luciferase
FEATURES             Location/Qualifiers
     source          1..5354
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    211..232
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(247..252,253..270,271..277)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    285..301
                     /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)."
     primer_bind     290..312
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     309..325
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     309..325
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             321..599
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSAQLTLTKGNKSWRTSRGGPVPNSPYSESYYARSLAVVL
                     QRRDWENPGVTQLNRLAAHPPFASWRNSEEARTDRPSQQLRSLNGEW"
     primer_bind     344..364
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        346..364
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(411..430)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(412..430)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(440..457)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(440..456)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(449..471)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             627..2276
                     /codon_start=1
                     /gene="luc+"
                     /product="firefly luciferase"
                     /label=luciferase
                     /note="enhanced luc+ version of the luciferase gene"
                     /translation="EDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAH
                     IEVDITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPA
                     NDIYNERELLNSMGISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSM
                     YTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGLPKGVALPHRTACVRFSHAR
                     DPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKI
                     QSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYG
                     LTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSG
                     YVNNPEATNALIDKDGWLHSGDIAYWDEDEHFFIVDRLKSLIKYKGYQVAPAELESILL
                     QHPNIFDAGVAGLPDDDAGELPAAVVVLEHGKTMTEKEIVDYVASQVTTAKKLRGGVVF
                     VDEVPKGLTGKLDARKIREILIKAKKGGKIAV"
     primer_bind     complement(690..708)
                     /label=LucNrev
                     /note="Firefly luciferase, reverse primer"
     polyA_signal    2317..2438
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2328..2347)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     2382..2401
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     complement(2686..2703)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2857..3445)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(2937..2956)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3616..4407,4408..4476))
                     /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     4239..4258
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4477..4581)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      4608..5063
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4695..4714)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     4905..4926
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     polyA_signal    5194..5242
                     /note="synthetic polyadenylation signal"
     misc_feature    5256..5347
                     /label=pause site
                     /note="RNA polymerase II transcriptional pause signal from 
                     the human alpha-2 globin gene"
     primer_bind     5296..5315
                     /label=RVprimer3
                     /note="pGL3 vector, forward primer"
ORIGIN
        1 ggtaccgagc tcttacgcgt gctagcccgg gctcgagatc tgacttctag agatccggca
       61 aaccccgccc agcgtcttgt cattggcgaa ttcgaacacg cagatgcagt cggggcggcg
      121 cggtccgagg tccacttcgc atattaaggt gacgcgtgtg gcctcgaaca cggagacgat
      181 ctggaaagcg ggcagtgagc gcaacgcaat taatgtgagt tagctcactc attaggcacc
      241 ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga gcggataaca
      301 atttcacaca ggaaacagct atgaccatga ttacgccaag cgcgcaatta accctcacta
      361 aagggaacaa aagctggagg acctcgaggg ggggcccggt acccaattcg ccctatagtg
      421 agtcgtatta cgcgcgctca ctggccgtcg ttttacaacg tcgtgactgg gaaaaccctg
      481 gcgttaccca acttaatcgc cttgcagcac atcccccttt cgccagctgg cgtaatagcg
      541 aagaggcccg caccgatcgc ccttcccaac agttgcgcag cctgaatggc gaatggtagg
      601 acgcgccctg tagcgtgggc gtctcagaag acgccaaaaa cataaagaaa ggcccggcgc
      661 cattctatcc gctggaagat ggaaccgctg gagagcaact gcataaggct atgaagagat
      721 acgccctggt tcctggaaca attgctttta cagatgcaca tatcgaggtg gacatcactt
      781 acgctgagta cttcgaaatg tccgttcggt tggcagaagc tatgaaacga tatgggctga
      841 atacaaatca cagaatcgtc gtatgcagtg aaaactctct tcaattcttt atgccggtgt
      901 tgggcgcgtt atttatcgga gttgcagttg cgcccgcgaa cgacatttat aatgaacgtg
      961 aattgctcaa cagtatgggc atttcgcagc ctaccgtggt gttcgtttcc aaaaaggggt
     1021 tgcaaaaaat tttgaacgtg caaaaaaagc tcccaatcat ccaaaaaatt attatcatgg
     1081 attctaaaac ggattaccag ggatttcagt cgatgtacac gttcgtcaca tctcatctac
     1141 ctcccggttt taatgaatac gattttgtgc cagagtcctt cgatagggac aagacaattg
     1201 cactgatcat gaactcctct ggatctactg gtctgcctaa aggtgtcgct ctgcctcata
     1261 gaactgcctg cgtgagattc tcgcatgcca gagatcctat ttttggcaat caaatcattc
     1321 cggatactgc gattttaagt gttgttccat tccatcacgg ttttggaatg tttactacac
     1381 tcggatattt gatatgtgga tttcgagtcg tcttaatgta tagatttgaa gaagagctgt
     1441 ttctgaggag ccttcaggat tacaagattc aaagtgcgct gctggtgcca accctattct
     1501 ccttcttcgc caaaagcact ctgattgaca aatacgattt atctaattta cacgaaattg
     1561 cttctggtgg cgctcccctc tctaaggaag tcggggaagc ggttgccaag aggttccatc
     1621 tgccaggtat caggcaagga tatgggctca ctgagactac atcagctatt ctgattacac
     1681 ccgaggggga tgataaaccg ggcgcggtcg gtaaagttgt tccatttttt gaagcgaagg
     1741 ttgtggatct ggataccggg aaaacgctgg gcgttaatca aagaggcgaa ctgtgtgtga
     1801 gaggtcctat gattatgtcc ggttatgtaa acaatccgga agcgaccaac gccttgattg
     1861 acaaggatgg atggctacat tctggagaca tagcttactg ggacgaagac gaacacttct
     1921 tcatcgttga ccgcctgaag tctctgatta agtacaaagg ctatcaggtg gctcccgctg
     1981 aattggaatc catcttgctc caacacccca acatcttcga cgcaggtgtc gcaggtcttc
     2041 ccgacgatga cgccggtgaa cttcccgccg ccgttgttgt tttggagcac ggaaagacga
     2101 tgacggaaaa agagatcgtg gattacgtcg ccagtcaagt aacaaccgcg aaaaagttgc
     2161 gcggaggagt tgtgtttgtg gacgaagtac cgaaaggtct taccggaaaa ctcgacgcaa
     2221 gaaaaatcag agagatcctc ataaaggcca agaagggcgg aaagatcgcc gtgtaattct
     2281 agagtcgggg cggccggccg cttcgagcag acatgataag atacattgat gagtttggac
     2341 aaaccacaac tagaatgcag tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
     2401 ctttatttgt aaccattata agctgcaata aacaagttaa caacaacaat tgcattcatt
     2461 ttatgtttca ggttcagggg gaggtgtggg aggtttttta aagcaagtaa aacctctaca
     2521 aatgtggtaa aatcgataag gatccgtcga ccgatgccct tgagagcctt caacccagtc
     2581 agctccttcc ggtgggcgcg gggcatgact atcgtcgccg cacttatgac tgtcttcttt
     2641 atcatgcaac tcgtaggaca ggtgccggca gcgctcttcc gcttcctcgc tcactgactc
     2701 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     2761 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     2821 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     2881 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     2941 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3001 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3061 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3121 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3181 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3241 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3301 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3361 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3421 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3481 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3541 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3601 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     3661 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     3721 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     3781 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     3841 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     3901 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     3961 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4021 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4081 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4141 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4201 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4261 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4321 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4381 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4441 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4501 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4561 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag
     4621 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgcta cacttgccag
     4681 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgt tcgccggctt
     4741 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtg ctttacggca
     4801 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccat cgccctgata
     4861 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggac tcttgttcca
     4921 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataag ggattttgcc
     4981 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacg cgaattttaa
     5041 caaaatatta acgcttacaa tttgccattc gccattcagg ctgcgcaact gttgggaagg
     5101 gcgatcggtg cgggcctctt cgctattacg ccagcccaag ctaccatgat aagtaagtaa
     5161 tattaaggta cgggaggtac ttggagcggc cgcaataaaa tatctttatt ttcattacat
     5221 ctgtgtgttg gttttttgtg tgaatcgata gtactaacat acgctctcca tcaaaacaaa
     5281 acgaaacaaa acaaactagc aaaataggct gtccccagtg caagtgcagg tgccagaaca
     5341 tttctctatc gata
//
