LOCUS       sequence-376043-        7972 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 7972)
  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 7972)
  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: SNW1
FEATURES             Location/Qualifiers
     source          1..7972
                     /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(2929..2953)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             3078..3119
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(3574..3591)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    3580..3804
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3937..3956)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    4088..4135
                     /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             4143..5165
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             4143..5165
                     /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    5295..5416
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(5332..5351)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     5386..5405
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(5465..5481)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5465..5481)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5478..5500)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5489..5505
                     /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(5513..5519,5520..5537,5538..5543))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5558..5579
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5696..5713)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5867..6455)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5947..5966)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6626..7417,7418..7486))
                     /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     7249..7268
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7487..7591)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(7666..7685)
                     /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 gttggcacca
     1321 tggcgctcac cagcttttta cctgcaccta ctcagctatc tcaggaccag cttgaggctg
     1381 aagaaaaggc aagatcccag agatcacggc agacctcact ggtctcctcc cgaagagaac
     1441 ctcccccgta cggataccgg aaaggctgga tacctcggtt attagaggat tttggagatg
     1501 gaggtgcttt tccagagatc catgtggccc agtatccact ggatatggga cgaaagaaaa
     1561 aaatgtcgaa tgcgctggcc attcaggtgg attctgaagg aaaaattaaa tatgatgcaa
     1621 ttgctcgaca aggacagtca aaagacaagg tcatttatag caaatacact gacctggttc
     1681 caaaggaggt tatgaatgca gatgatccag acctgcaaag gcccgatgaa gaagctatta
     1741 aagagataac agaaaagaca agagtagcct tagaaaaatc tgtatcacag aaggtcgccg
     1801 cagccatgcc agttcgagca gctgacaaat tggctcctgc tcagtatatc cgatacacac
     1861 catctcagca aggagtggca ttcaactctg gagctaaaca gagggttatt cggatggtag
     1921 aaatgcagaa agatccaatg gagcctccaa ggttcaagat taataagaaa attccccggg
     1981 gaccaccttc tcctcctgcg cctgtcatgc attctcctag ccgaaagatg actgtaaagg
     2041 aacaacaaga gtggaagatt cctccttgta tttctaactg gaaaaatgca aagggttata
     2101 caattccatt agacaaacgt ctggctgctg atggaagagg actacagaca gtacacataa
     2161 atgaaaattt cgccaaattg gcagaagccc tctacattgc tgatcggaag gctcgtgaag
     2221 ctgtggaaat gcgtgcccaa gtagagagaa aaatggctca gaaagaaaag gaaaaacatg
     2281 aagagaaact tagagaaatg gcccagaaag ccagggaaag aagagctggg atcaaaactc
     2341 atgtggaaaa agaggatggg gaggcacgtg agagggatga aatccggcat gacaggcgaa
     2401 aagagagaca gcatgaccgg aatctttcca gggcagctcc tgataagagg tcgaaacttc
     2461 agagaaatga aaatcgggat atcagtgaag ttattgctct cggtgttcct aatcctcgga
     2521 cttccaatga agttcagtat gaccaaaggc tcttcaacca atccaagggt atggacagtg
     2581 gatttgcagg tggagaagat gaaatttata atgtttatga tcaagcctgg agaggtggta
     2641 aagatatggc ccagagtatt tataggccca gtaaaaatct ggacaaggac atgtatggtg
     2701 atgacctaga agccagaata aagaccaaca gatttgttcc cgacaaggag ttttctggtt
     2761 cagaccgtag acagagaggc cgagaaggac cagttcagtt tgaggaagat ccttttggtt
     2821 tggacaagtt tttggaagaa gccaaacagc atggtggctc taaaagaccc tcagatagca
     2881 gccgccccaa ggaacacgag catgaaggca agaagaggag gaaggaattg ccaactttct
     2941 tgtacaaagt ggttgatatc cagcacagtg gcggccgctc gagtctagag ggcccgcggt
     3001 tcgaaaccag cggaggcggc ggaagcgggg gaggaggcag cggcggaggc ggcagcgggg
     3061 gcggcggaag cgctaccggt aagcctatcc ctaaccctct cctcggtctc gattctacgc
     3121 gtaccggtat ggcttctaac tttactcagt tcgttctcgt cgacaatggc ggaactggcg
     3181 acgtgactgt cgccccaagc aacttcgcta acggggtcgc tgaatggatc agctctaact
     3241 cgcgatcaca ggcttacaaa gtaacctgta gcgttcgtca gagctctgcg cagaatcgca
     3301 aatacaccat caaagtcgag gtgcctaaag tggcaaccca gactgttggt ggtgaagagc
     3361 ttcctgtagc cggatggaga tcttacttaa atatggaact aaccattcca attttcgcca
     3421 cgaattccga ctgcgagctt attgttaagg caatgcaagg tctcctaaaa gatggaaacc
     3481 cgattccctc ggccatcgcg gccaactccg gcatctacgg aggtggaggt ggatctcgta
     3541 cgggttagta atgagtttaa acccgctgat cagcctcgac tgtgccttct agttgccagc
     3601 catctgttgt ttgcccctcc cccgtgcctt ccttgaccct ggaaggtgcc actcccactg
     3661 tcctttccta ataaaatgag gaaattgcat cgcattgtct gagtaggtgt cattctattc
     3721 tggggggtgg ggtggggcag gacagcaagg gggaggattg ggaagacaat agcaggcatg
     3781 ctggggatgc ggtgggctct atggcttctg aggcggaaag aaccagctgg ggctctaggg
     3841 ggtatcccca cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca
     3901 gcgtgaccgc tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct
     3961 ttctcgccac gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt
     4021 tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
     4081 gtacctagaa gttcctattc cgaagttcct attctctaga aagtatagga acttccttgg
     4141 ccaaaaagcc tgaactcacc gcgacgtctg tcgagaagtt tctgatcgaa aagttcgaca
     4201 gcgtctccga cctgatgcag ctctcggagg gcgaagaatc tcgtgctttc agcttcgatg
     4261 taggagggcg tggatatgtc ctgcgggtaa atagctgcgc cgatggtttc tacaaagatc
     4321 gttatgttta tcggcacttt gcatcggccg cgctcccgat tccggaagtg cttgacattg
     4381 gggaattcag cgagagcctg acctattgca tctcccgccg tgcacagggt gtcacgttgc
     4441 aagacctgcc tgaaaccgaa ctgcccgctg ttctgcagcc ggtcgcggag gccatggatg
     4501 cgatcgctgc ggccgatctt agccagacga gcgggttcgg cccattcgga ccgcaaggaa
     4561 tcggtcaata cactacatgg cgtgatttca tatgcgcgat tgctgatccc catgtgtatc
     4621 actggcaaac tgtgatggac gacaccgtca gtgcgtccgt cgcgcaggct ctcgatgagc
     4681 tgatgctttg ggccgaggac tgccccgaag tccggcacct cgtgcacgcg gatttcggct
     4741 ccaacaatgt cctgacggac aatggccgca taacagcggt cattgactgg agcgaggcga
     4801 tgttcgggga ttcccaatac gaggtcgcca acatcttctt ctggaggccg tggttggctt
     4861 gtatggagca gcagacgcgc tacttcgagc ggaggcatcc ggagcttgca ggatcgccgc
     4921 ggctccgggc gtatatgctc cgcattggtc ttgaccaact ctatcagagc ttggttgacg
     4981 gcaatttcga tgatgcagct tgggcgcagg gtcgatgcga cgcaatcgtc cgatccggag
     5041 ccgggactgt cgggcgtaca caaatcgccc gcagaagcgc ggccgtctgg accgatggct
     5101 gtgtagaagt actcgccgat agtggaaacc gacgccccag cactcgtccg agggcaaagg
     5161 aatagcacgt actacgagat ttcgattcca ccgccgcctt ctatgaaagg ttgggcttcg
     5221 gaatcgtttt ccgggacgcc ggctggatga tcctccagcg cggggatctc atgctggagt
     5281 tcttcgccca ccccaacttg tttattgcag cttataatgg ttacaaataa agcaatagca
     5341 tcacaaattt cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac
     5401 tcatcaatgt atcttatcat gtctgtatac cgtcgacctc tagctagagc ttggcgtaat
     5461 catggtcata gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac
     5521 gagccggaag cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa
     5581 ttgcgttgcg ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat
     5641 gaatcggcca acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc
     5701 tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg
     5761 cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag
     5821 gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc
     5881 gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag
     5941 gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga
     6001 ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc
     6061 atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg
     6121 tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt
     6181 ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca
     6241 gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca
     6301 ctagaagaac agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag
     6361 ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca
     6421 agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg
     6481 ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa
     6541 aaaggatctt cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta
     6601 tatatgagta aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag
     6661 cgatctgtct atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga
     6721 tacgggaggg cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac
     6781 cggctccaga tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc
     6841 ctgcaacttt atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta
     6901 gttcgccagt taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac
     6961 gctcgtcgtt tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat
     7021 gatcccccat gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa
     7081 gtaagttggc cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg
     7141 tcatgccatc cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag
     7201 aatagtgtat gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc
     7261 cacatagcag aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct
     7321 caaggatctt accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat
     7381 cttcagcatc ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg
     7441 ccgcaaaaaa gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc
     7501 aatattattg aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta
     7561 tttagaaaaa taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg
     7621 tcgacggatc gggagatctc ccgatcccct atggtgcact ctcagtacaa tctgctctga
     7681 tgccgcatag ttaagccagt atctgctccc tgcttgtgtg ttggaggtcg ctgagtagtg
     7741 cgcgagcaaa atttaagcta caacaaggca aggcttgacc gacaattgca tgaagaatct
     7801 gcttagggtt aggcgttttg cgctgcttcg cgatgtacgg gccagatata cgcgttgaca
     7861 ttgattattg actaggcttt tgcaaaaagc tttgcaaaga tggataaagt tttaaacaga
     7921 gaggaatctt tgcagctaat ggaccttcta ggtcttgaaa ggagtgggaa tt
//
