LOCUS       sequence-343587-        6997 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For expressing siRNA-resistant frog ePAB in human cell lines via 
            transfection.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6997)
  AUTHORS   Xiang K, Bartel DP
  TITLE     The molecular basis of coupling between poly(A)-tail length and 
            translational efficiency.
  JOURNAL   Elife. 2021 Jul 2;10. pii: 66493. doi: 10.7554/eLife.66493.
  PUBMED    34213414
REFERENCE   2  (bases 1 to 6997)
  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: "Elife."; 
            date: "2021-07-2"; volume: "10. pii: 66493. doi"; pages: " 
            10.7554/eLife.66493"
COMMENT     Sequence Label: C205
FEATURES             Location/Qualifiers
     source          1..6997
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     enhancer        1..380
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        381..584
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     535..555
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    586..604
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    607..625
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     635..659
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      753..762
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             762..785
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(2715..2732)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2721..2945
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3078..3097)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3229..3276
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3284..4306
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3284..4306
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4436..4557
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4473..4492)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4527..4546
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4606..4622)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4606..4622)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4619..4641)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4630..4646
                     /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(4654..4660,4661..4678,4679..4684))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4699..4720
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4837..4854)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5008..5596)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5088..5107)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5767..6558,6559..6627))
                     /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     6390..6409
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6628..6732)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6807..6826)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 gacattgatt attgactagt tattaatagt aatcaattac ggggtcatta gttcatagcc
       61 catatatgga gttccgcgtt acataactta cggtaaatgg cccgcctggc tgaccgccca
      121 acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga
      181 ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc
      241 aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct
      301 ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat
      361 tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc
      421 ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt
      481 ggcaccaaaa tcaacgggac tttccaaaat gtcgtaacaa ctccgcccca ttgacgcaaa
      541 tgggcggtag gcgtgtacgg tgggaggtct atataagcag agctctccct atcagtgata
      601 gagatctccc tatcagtgat agagatcgtc gacgagctcg tttagtgaac cgtcagatcg
      661 cctggagacg ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc
      721 tccggactct agcgtttaaa cttaagcttg ccgccaccat ggattacaag gatgacgacg
      781 ataagatgaa cgcaaccaga gccgaatacc cgctggcatc tctttatata ggggacctcc
      841 accccgacgt gacggaagcg atgctgtatg agaagttttc tccagcgggg cccatcatgt
      901 ctataagagt gtgtcgagat attgctactc gccggtccct tggctatgcc tacattaatt
      961 tccagcagcc ggctgatgct gagcgggcct tggatacaat gaattttgag gttattaaag
     1021 gcaggccaat ccgaatcatg tggtcccaga gagatccggg ccttcgaaaa tcgggtgtag
     1081 ggaatgtatt tattaaaaat ttagacgact ccatcgacaa caaagccctg tatgacactt
     1141 tctcagcgtt tggagatatt ttatcttgca aagttgtgtg cgatgaatac ggatctcgag
     1201 gttatggttt cgtacatttt gagacccaag aagctgccaa tcgagcgatt cagactatga
     1261 atgggatgtt gctcaacgac cgcaaagtgt ttgtgggcca ttttaaatca cggagggaaa
     1321 gagaattaga atatggggca aaagtcatgg aatttaccaa cgtgtacatc aagaacttcg
     1381 gggaggatat ggacgataaa agacttaaag aaatattttc tgcctttgga aatacgttaa
     1441 gcgttaaagt aatgatggat aatagtggtc gatcccgggg ttttggtttt gttaattatg
     1501 gaaatcacga agaagcacaa aaagcagtta ccgaaatgaa tggtaaagaa gtaaacggtc
     1561 gaatggttta cgtaggcaga gctcagaaga ggatagaacg gcagggcgag ttaaaacgca
     1621 agttcgaaca aatcaagcaa gaacgaatca accgctatca gggtgttaat ttgtatgtta
     1681 aaaacctgga cgatggtatc gatgatgacc ggctcagaaa ggagttctcg ccgtatggta
     1741 ccattaccag tacaaaggta atgactgaag gaggccatag taaaggcttt ggttttgttt
     1801 gcttctcctc cccagaagaa gccactaaag cagtcacaga aatgaacggg aggatagtca
     1861 gtacgaagcc tctatatgtt gcacttgcac aaaggaagga agaacggaag gcaattctaa
     1921 ccaatcagta catgcagagg ctagccacta tgagggccat gcctggtcct ctccttgggt
     1981 ccttccagca acctgctaat tatttcttgc ctacaatgcc acagccttca aacagagcat
     2041 tttatagtcc aaatcctgtt gctcctgtcc gtccagctcc tcagtgggct tcccaccagt
     2101 ccaggcctcc acagtatcaa ccaccagccc cattaatgag ggcggtgcca ccaagaagga
     2161 tgcattcaaa tatcagtaca atgaagcagg catcaacaca agtacccagg gtgcctctac
     2221 agagtcaaag agtagcaaac attggcactc aaacagctgg agctcgggcc caagtaaacg
     2281 cgtccataat gcgcgcaatg ccacattaca agtattcctg tggcgtgagg aacgtgcagc
     2341 ctataggaag ctctgctcat ctgcaacagg tactggaacc tgcagttctg atgcaaggac
     2401 aagaaccgct aactgcatct ctgcttgcag ctgccccact gcaggaacag aaacaaatac
     2461 tcggggaacg tatttacccg ttaatacatg aaatgcatcc gacgttagcc ggaaagataa
     2521 cgggaatgct gctggaaatt gataactctg agctactgca tatgctggag tcaccagagt
     2581 ctctgcattc aaaggtggaa gaggcagtgg cggtcctgca agcccaccaa gccaaggaaa
     2641 gtgctccgaa aagtgctccg cagtctctga tctgacgctc gagtctagag ggcccgttta
     2701 aacccgctga tcagcctcga ctgtgccttc tagttgccag ccatctgttg tttgcccctc
     2761 ccccgtgcct tccttgaccc tggaaggtgc cactcccact gtcctttcct aataaaatga
     2821 ggaaattgca tcgcattgtc tgagtaggtg tcattctatt ctggggggtg gggtggggca
     2881 ggacagcaag ggggaggatt gggaagacaa tagcaggcat gctggggatg cggtgggctc
     2941 tatggcttct gaggcggaaa gaaccagctg gggctctagg gggtatcccc acgcgccctg
     3001 tagcggcgca ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc
     3061 cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg
     3121 ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta gtgctttacg
     3181 gcacctcgac cccaaaaaac ttgattaggg tgatggttca cgtacctaga agttcctatt
     3241 ccgaagttcc tattctctag aaagtatagg aacttccttg gccaaaaagc ctgaactcac
     3301 cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac agcgtctccg acctgatgca
     3361 gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat gtaggagggc gtggatatgt
     3421 cctgcgggta aatagctgcg ccgatggttt ctacaaagat cgttatgttt atcggcactt
     3481 tgcatcggcc gcgctcccga ttccggaagt gcttgacatt ggggaattca gcgagagcct
     3541 gacctattgc atctcccgcc gtgcacaggg tgtcacgttg caagacctgc ctgaaaccga
     3601 actgcccgct gttctgcagc cggtcgcgga ggccatggat gcgatcgctg cggccgatct
     3661 tagccagacg agcgggttcg gcccattcgg accgcaagga atcggtcaat acactacatg
     3721 gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat cactggcaaa ctgtgatgga
     3781 cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag ctgatgcttt gggccgagga
     3841 ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc tccaacaatg tcctgacgga
     3901 caatggccgc ataacagcgg tcattgactg gagcgaggcg atgttcgggg attcccaata
     3961 cgaggtcgcc aacatcttct tctggaggcc gtggttggct tgtatggagc agcagacgcg
     4021 ctacttcgag cggaggcatc cggagcttgc aggatcgccg cggctccggg cgtatatgct
     4081 ccgcattggt cttgaccaac tctatcagag cttggttgac ggcaatttcg atgatgcagc
     4141 ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga gccgggactg tcgggcgtac
     4201 acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc tgtgtagaag tactcgccga
     4261 tagtggaaac cgacgcccca gcactcgtcc gagggcaaag gaatagcacg tactacgaga
     4321 tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt tccgggacgc
     4381 cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc accccaactt
     4441 gtttattgca gcttataatg gttacaaata aagcaatagc atcacaaatt tcacaaataa
     4501 agcatttttt tcactgcatt ctagttgtgg tttgtccaaa ctcatcaatg tatcttatca
     4561 tgtctgtata ccgtcgacct ctagctagag cttggcgtaa tcatggtcat agctgtttcc
     4621 tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggaa gcataaagtg
     4681 taaagcctgg ggtgcctaat gagtgagcta actcacatta attgcgttgc gctcactgcc
     4741 cgctttccag tcgggaaacc tgtcgtgcca gctgcattaa tgaatcggcc aacgcgcggg
     4801 gagaggcggt ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc
     4861 ggtcgttcgg ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac
     4921 agaatcaggg gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa
     4981 ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca
     5041 caaaaatcga cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc
     5101 gtttccccct ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata
     5161 cctgtccgcc tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta
     5221 tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca
     5281 gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga
     5341 cttatcgcca ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg
     5401 tgctacagag ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg
     5461 tatctgcgct ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg
     5521 caaacaaacc accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag
     5581 aaaaaaagga tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa
     5641 cgaaaactca cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat
     5701 ccttttaaat taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc
     5761 tgacagttac caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc
     5821 atccatagtt gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc
     5881 tggccccagt gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc
     5941 aataaaccag ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc
     6001 catccagtct attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt
     6061 gcgcaacgtt gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
     6121 ttcattcagc tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa
     6181 aaaagcggtt agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt
     6241 atcactcatg gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg
     6301 cttttctgtg actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc
     6361 gagttgctct tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa
     6421 agtgctcatc attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
     6481 gagatccagt tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt
     6541 caccagcgtt tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag
     6601 ggcgacacgg aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta
     6661 tcagggttat tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat
     6721 aggggttccg cgcacatttc cccgaaaagt gccacctgac gtcgacggat cgggagatct
     6781 cccgatcccc tatggtgcac tctcagtaca atctgctctg atgccgcata gttaagccag
     6841 tatctgctcc ctgcttgtgt gttggaggtc gctgagtagt gcgcgagcaa aatttaagct
     6901 acaacaaggc aaggcttgac cgacaattgc atgaagaatc tgcttagggt taggcgtttt
     6961 gcgctgcttc gcgatgtacg ggccagatat acgcgtt
//
