LOCUS       sequence-376940-        8066 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  For use in RBP tethering screen.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8066)
  AUTHORS   Luo EC, Nathanson JL, Tan FE, Schwartz JL, Schmok JC, Shankar A, 
            Markmiller S, Yee BA, Sathe S, Pratt GA, Scaletta DB, Ha Y, Hill DE,
            Aigner S, Yeo GW
  TITLE     Large-scale tethered function assays identify factors that regulate 
            mRNA stability and translation.
  JOURNAL   Nat Struct Mol Biol. 2020 Oct;27(10):989-1000. doi: 
            10.1038/s41594-020-0477-6. Epub 2020 Aug 17.
  PUBMED    32807991
REFERENCE   2  (bases 1 to 8066)
  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"; doi: 
            "10.1038/s41594-020-0477-6"; journalName: "Nat Struct Mol Biol"; 
            date: "2020-10"; volume: "27"; issue: "10"; pages: "989-1000"
COMMENT     Sequence Label: NSUN2
FEATURES             Location/Qualifiers
     source          1..8066
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..1179
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          232..1170
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1127..1147
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1196..1215
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1196..1214
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1293..1317
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(3023..3047)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3172..3213
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3668..3685)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3674..3898
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(4031..4050)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4182..4229
                     /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             4237..5259
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4237..5259
                     /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    5389..5510
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5426..5445)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5480..5499
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5559..5575)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5559..5575)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5572..5594)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5583..5599
                     /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(5607..5613,5614..5631,5632..5637))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5652..5673
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5790..5807)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5961..6549)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6041..6060)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6720..7511,7512..7580))
                     /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     7343..7362
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7581..7685)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7760..7779)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 ggctccggtg cccgtcagtg ggcagagcgc acatcgccca cagtccccga gaagttgggg
       61 ggaggggtcg gcaattgaac cggtgcctag agaaggtggc gcggggtaaa ctgggaaagt
      121 gatgtcgtgt actggctccg cctttttccc gagggtgggg gagaaccgta tataagtgca
      181 gtagtcgccg tgaacgttct ttttcgcaac gggtttgccg ccagaacaca ggtaagtgcc
      241 gtgtgtggtt cccgcgggcc tggcctcttt acgggttatg gcccttgcgt gccttgaatt
      301 acttccacct ggctgcagta cgtgattctt gatcccgagc ttcgggttgg aagtgggtgg
      361 gagagttcga ggccttgcgc ttaaggagcc ccttcgcctc gtgcttgagt tgaggcctgg
      421 cctgggcgct ggggccgccg cgtgcgaatc tggtggcacc ttcgcgcctg tctcgctgct
      481 ttcgataagt ctctagccat ttaaaatttt tgatgacctg ctgcgacgct ttttttctgg
      541 caagatagtc ttgtaaatgc gggccaagat ctgcacactg gtatttcggt ttttggggcc
      601 gcgggcggcg acggggcccg tgcgtcccag cgcacatgtt cggcgaggcg gggcctgcga
      661 gcgcggccac cgagaatcgg acgggggtag tctcaagctg gccggcctgc tctggtgcct
      721 ggcctcgcgc cgccgtgtat cgccccgccc tgggcggcaa ggctggcccg gtcggcacca
      781 gttgcgtgag cggaaagatg gccgcttccc ggccctgctg cagggagctc aaaatggagg
      841 acgcggcgct cgggagagcg ggcgggtgag tcacccacac aaaggaaaag ggcctttccg
      901 tcctcagccg tcgcttcatg tgactccacg gagtaccggg cgccgtccag gcacctcgat
      961 tagttctcga gcttttggag tacgtcgtct ttaggttggg gggaggggtt ttatgcgatg
     1021 gagtttcccc acactgagtg ggtggagact gaagttaggc cagcttggca cttgatgtaa
     1081 ttctccttgg aatttgccct ttttgagttt ggatcttggt tcattctcaa gcctcagaca
     1141 gtggttcaaa gtttttttct tccatttcag gtgtcgtgag gaattagctt ggtactaata
     1201 cgactcacta tagggagacc caagctggct aggtaagctt ggtaccgagc tcggatccac
     1261 tagtccagtg tggtggaatt ctgcagatat caacaagttt gtacaaaaaa gcaggctgcc
     1321 accatgaatg tcccctttcc agagggattt gttattgcga atgatgtgga caacaagcgc
     1381 tgctacctgc tcgtccatca agccaagagg ctgagcagcc cctgcatcat ggtggtcaac
     1441 catgatgcct ccagcatacc caggctccag atagatgtgg acggcaggaa agagatcctc
     1501 ttctatgatc gaattttatg tgatgtccct tgcagtggag acggcactat gagaaaaaac
     1561 attgatgttt ggaaaaagtg gaccacctta aatagcttgc agctacatgg cttacagctg
     1621 cggattgcaa cacgcggggc tgaacagctg gctgaaggtg gaaggatggt gtattccacg
     1681 tgttcactaa accctattga ggatgaagca gtcatagcat ctttactgga aaaaagtgaa
     1741 ggtgctttgg agcttgctga tgtgtctaat gaactgccag ggctgaagtg gatgcctgga
     1801 atcacacagt ggaaggtaat gacgaaagat gggcagtggt ttacagactg ggacgctgtt
     1861 cctcacagca gacacaccca gatccgacct accatgttcc ctccgaagga cccagaaaag
     1921 ctgcaggcca tgcacctgga gcgatgcctt aggatattac cccatcatca gaatactgga
     1981 gggttttttg tggcagtatt ggtgaaaaaa tcttcaatgc cgtggaataa acgtcagcca
     2041 aagcttcagg gtaaatctgc agagaccaga gaaagcacac agctgagccc tgcagatctc
     2101 acagaaggga aacccacaga tccctctaag ctggaaagtc cgtcattcac aggaactggt
     2161 gacacagaaa tagctcatgc aactgaggat ttagagaata atggcagtaa gaaagatggc
     2221 gtgtgtggtc ctcctccatc aaagaaaatg aagttatttg gatttaaaga agatccattt
     2281 gtatttattc ctgaagatga cccattattt ccacctattg agaaatttta tgctttggat
     2341 ccttcattcc caaggatgaa tttgttaact cggactacag aagggaagaa aaggcagctc
     2401 tacatggttt ctaaggagtt gcggaatgtg ctgctgaata acagtgagaa gatgaaggtt
     2461 attaacacgg ggatcaaagt ctggtgtaga aataacagcg gtgaagagtt tgactgtgct
     2521 ttccggctgg cacaggaggg aatatataca ttgtatccat ttattaactc aagaattatt
     2581 actgtatcaa tggaagatgt taagatactg ttgacccagg aaaatccctt ttttagaaaa
     2641 ctcagcagtg agacctacag tcaagcaaag gacctggcaa agggaagcat cgtgctgaag
     2701 tatgaaccag attctgcgaa tccagacgct ctgcagtgtc ccatcgtctt atgcggatgg
     2761 cggggaaagg cctccattcg aacttttgtg cccaagaatg aacggcttca ttatctcagg
     2821 atgatggggc tggaggtatt gggagaaaag aagaaggaag gggttatcct cacaaatgag
     2881 agtgcagcca gcaccggaca gccagacaat gacgtgactg agggacagag agcaggagag
     2941 cccaacagcc cagatgcaga agaggccaac agtccagacg tgacagcagg ctgtgacccg
     3001 gcgggggtcc atccaccccg ggacccagct ttcttgtaca aagtggttga tatccagcac
     3061 agtggcggcc gctcgagtct agagggcccg cggttcgaaa ccagcggagg cggcggaagc
     3121 gggggaggag gcagcggcgg aggcggcagc gggggcggcg gaagcgctac cggtaagcct
     3181 atccctaacc ctctcctcgg tctcgattct acgcgtaccg gtatggcttc taactttact
     3241 cagttcgttc tcgtcgacaa tggcggaact ggcgacgtga ctgtcgcccc aagcaacttc
     3301 gctaacgggg tcgctgaatg gatcagctct aactcgcgat cacaggctta caaagtaacc
     3361 tgtagcgttc gtcagagctc tgcgcagaat cgcaaataca ccatcaaagt cgaggtgcct
     3421 aaagtggcaa cccagactgt tggtggtgaa gagcttcctg tagccggatg gagatcttac
     3481 ttaaatatgg aactaaccat tccaattttc gccacgaatt ccgactgcga gcttattgtt
     3541 aaggcaatgc aaggtctcct aaaagatgga aacccgattc cctcggccat cgcggccaac
     3601 tccggcatct acggaggtgg aggtggatct cgtacgggtt agtaatgagt ttaaacccgc
     3661 tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc ctcccccgtg
     3721 ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa tgaggaaatt
     3781 gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg gcaggacagc
     3841 aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg ctctatggct
     3901 tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc ctgtagcggc
     3961 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     4021 ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     4081 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     4141 gaccccaaaa aacttgatta gggtgatggt tcacgtacct agaagttcct attccgaagt
     4201 tcctattctc tagaaagtat aggaacttcc ttggccaaaa agcctgaact caccgcgacg
     4261 tctgtcgaga agtttctgat cgaaaagttc gacagcgtct ccgacctgat gcagctctcg
     4321 gagggcgaag aatctcgtgc tttcagcttc gatgtaggag ggcgtggata tgtcctgcgg
     4381 gtaaatagct gcgccgatgg tttctacaaa gatcgttatg tttatcggca ctttgcatcg
     4441 gccgcgctcc cgattccgga agtgcttgac attggggaat tcagcgagag cctgacctat
     4501 tgcatctccc gccgtgcaca gggtgtcacg ttgcaagacc tgcctgaaac cgaactgccc
     4561 gctgttctgc agccggtcgc ggaggccatg gatgcgatcg ctgcggccga tcttagccag
     4621 acgagcgggt tcggcccatt cggaccgcaa ggaatcggtc aatacactac atggcgtgat
     4681 ttcatatgcg cgattgctga tccccatgtg tatcactggc aaactgtgat ggacgacacc
     4741 gtcagtgcgt ccgtcgcgca ggctctcgat gagctgatgc tttgggccga ggactgcccc
     4801 gaagtccggc acctcgtgca cgcggatttc ggctccaaca atgtcctgac ggacaatggc
     4861 cgcataacag cggtcattga ctggagcgag gcgatgttcg gggattccca atacgaggtc
     4921 gccaacatct tcttctggag gccgtggttg gcttgtatgg agcagcagac gcgctacttc
     4981 gagcggaggc atccggagct tgcaggatcg ccgcggctcc gggcgtatat gctccgcatt
     5041 ggtcttgacc aactctatca gagcttggtt gacggcaatt tcgatgatgc agcttgggcg
     5101 cagggtcgat gcgacgcaat cgtccgatcc ggagccggga ctgtcgggcg tacacaaatc
     5161 gcccgcagaa gcgcggccgt ctggaccgat ggctgtgtag aagtactcgc cgatagtgga
     5221 aaccgacgcc ccagcactcg tccgagggca aaggaatagc acgtactacg agatttcgat
     5281 tccaccgccg ccttctatga aaggttgggc ttcggaatcg ttttccggga cgccggctgg
     5341 atgatcctcc agcgcgggga tctcatgctg gagttcttcg cccaccccaa cttgtttatt
     5401 gcagcttata atggttacaa ataaagcaat agcatcacaa atttcacaaa taaagcattt
     5461 ttttcactgc attctagttg tggtttgtcc aaactcatca atgtatctta tcatgtctgt
     5521 ataccgtcga cctctagcta gagcttggcg taatcatggt catagctgtt tcctgtgtga
     5581 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     5641 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     5701 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     5761 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5821 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5881 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5941 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     6001 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     6061 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     6121 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     6181 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     6241 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     6301 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     6361 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     6421 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     6481 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     6541 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     6601 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     6661 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     6721 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6781 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6841 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6901 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6961 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     7021 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     7081 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     7141 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     7201 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     7261 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     7321 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     7381 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     7441 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     7501 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     7561 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     7621 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     7681 ccgcgcacat ttccccgaaa agtgccacct gacgtcgacg gatcgggaga tctcccgatc
     7741 ccctatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatctgc
     7801 tccctgcttg tgtgttggag gtcgctgagt agtgcgcgag caaaatttaa gctacaacaa
     7861 ggcaaggctt gaccgacaat tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc
     7921 ttcgcgatgt acgggccaga tatacgcgtt gacattgatt attgactagg cttttgcaaa
     7981 aagctttgca aagatggata aagttttaaa cagagaggaa tctttgcagc taatggacct
     8041 tctaggtctt gaaaggagtg ggaatt
//
