LOCUS       sequence-376026-        6782 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 6782)
  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 6782)
  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: CRABP2
FEATURES             Location/Qualifiers
     source          1..6782
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        187..1365
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          418..1356
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1313..1333
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1382..1401
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1382..1400
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    1479..1503
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     protein_bind    complement(1925..1949)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     CDS             2074..2115
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     primer_bind     complement(2570..2587)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2576..2800
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(2933..2952)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3084..3131
                     /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             3139..4161
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3139..4161
                     /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    4291..4412
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4328..4347)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4382..4401
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4461..4477)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4461..4477)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4474..4496)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4485..4501
                     /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(4509..4515,4516..4533,4534..4539))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4554..4575
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4692..4709)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4863..5451)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4943..4962)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5622..6413,6414..6482))
                     /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     6245..6264
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6483..6587)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6662..6681)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
ORIGIN
        1 tgcatgaaga atctgcttag ggttaggcgt tttgcgctgc ttcgcgatgt acgggccaga
       61 tatacgcgtt gacattgatt attgactagg cttttgcaaa aagctttgca aagatggata
      121 aagttttaaa cagagaggaa tctttgcagc taatggacct tctaggtctt gaaaggagtg
      181 ggaattggct ccggtgcccg tcagtgggca gagcgcacat cgcccacagt ccccgagaag
      241 ttggggggag gggtcggcaa ttgaaccggt gcctagagaa ggtggcgcgg ggtaaactgg
      301 gaaagtgatg tcgtgtactg gctccgcctt tttcccgagg gtgggggaga accgtatata
      361 agtgcagtag tcgccgtgaa cgttcttttt cgcaacgggt ttgccgccag aacacaggta
      421 agtgccgtgt gtggttcccg cgggcctggc ctctttacgg gttatggccc ttgcgtgcct
      481 tgaattactt ccacctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt
      541 gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag
      601 gcctggcctg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc
      661 gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt
      721 ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt
      781 ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc
      841 ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg
      901 gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg
      961 gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa
     1021 tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc
     1081 tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac
     1141 ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat
     1201 gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg
     1261 atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct
     1321 cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgaggaat tagcttggta
     1381 ctaatacgac tcactatagg gagacccaag ctggctaggt aagcttggta ccgagctcgg
     1441 atccactagt ccagtgtggt ggaattctgc agatatcaac aagtttgtac aaaaaagcag
     1501 gctgccacca tgcccaactt ctctggcaac tggaaaatca tccgatcgga aaacttcgag
     1561 gaattgctca aagtgctggg ggtgaatgtg atgctgagga agattgctgt ggctgcagcg
     1621 tccaagccag cagtggagat caaacaggag ggagacactt tctacatcaa aacctccacc
     1681 accgtgcgca ccacagagat taacttcaag gttggggagg agtttgagga gcagactgtg
     1741 gatgggaggc cctgtaagag cctggtgaaa tgggagagtg agaataaaat ggtctgtgag
     1801 cagaagctcc tgaagggaga gggccccaag acctcgtgga ccagagaact gaccaacgat
     1861 ggggaactga tcctgaccat gacggcggat gacgttgtgt gcaccagggt ctccgtccga
     1921 aaggacccag ctttcttgta caaagtggtt gatatccagc acagtggcgg ccgctcgagt
     1981 ctagagggcc cgcggttcga aaccagcgga ggcggcggaa gcgggggagg aggcagcggc
     2041 ggaggcggca gcgggggcgg cggaagcgct accggtaagc ctatccctaa ccctctcctc
     2101 ggtctcgatt ctacgcgtac cggtatggct tctaacttta ctcagttcgt tctcgtcgac
     2161 aatggcggaa ctggcgacgt gactgtcgcc ccaagcaact tcgctaacgg ggtcgctgaa
     2221 tggatcagct ctaactcgcg atcacaggct tacaaagtaa cctgtagcgt tcgtcagagc
     2281 tctgcgcaga atcgcaaata caccatcaaa gtcgaggtgc ctaaagtggc aacccagact
     2341 gttggtggtg aagagcttcc tgtagccgga tggagatctt acttaaatat ggaactaacc
     2401 attccaattt tcgccacgaa ttccgactgc gagcttattg ttaaggcaat gcaaggtctc
     2461 ctaaaagatg gaaacccgat tccctcggcc atcgcggcca actccggcat ctacggaggt
     2521 ggaggtggat ctcgtacggg ttagtaatga gtttaaaccc gctgatcagc ctcgactgtg
     2581 ccttctagtt gccagccatc tgttgtttgc ccctcccccg tgccttcctt gaccctggaa
     2641 ggtgccactc ccactgtcct ttcctaataa aatgaggaaa ttgcatcgca ttgtctgagt
     2701 aggtgtcatt ctattctggg gggtggggtg gggcaggaca gcaaggggga ggattgggaa
     2761 gacaatagca ggcatgctgg ggatgcggtg ggctctatgg cttctgaggc ggaaagaacc
     2821 agctggggct ctagggggta tccccacgcg ccctgtagcg gcgcattaag cgcggcgggt
     2881 gtggtggtta cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc
     2941 gctttcttcc cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg
     3001 gggctccctt tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat
     3061 tagggtgatg gttcacgtac ctagaagttc ctattccgaa gttcctattc tctagaaagt
     3121 ataggaactt ccttggccaa aaagcctgaa ctcaccgcga cgtctgtcga gaagtttctg
     3181 atcgaaaagt tcgacagcgt ctccgacctg atgcagctct cggagggcga agaatctcgt
     3241 gctttcagct tcgatgtagg agggcgtgga tatgtcctgc gggtaaatag ctgcgccgat
     3301 ggtttctaca aagatcgtta tgtttatcgg cactttgcat cggccgcgct cccgattccg
     3361 gaagtgcttg acattgggga attcagcgag agcctgacct attgcatctc ccgccgtgca
     3421 cagggtgtca cgttgcaaga cctgcctgaa accgaactgc ccgctgttct gcagccggtc
     3481 gcggaggcca tggatgcgat cgctgcggcc gatcttagcc agacgagcgg gttcggccca
     3541 ttcggaccgc aaggaatcgg tcaatacact acatggcgtg atttcatatg cgcgattgct
     3601 gatccccatg tgtatcactg gcaaactgtg atggacgaca ccgtcagtgc gtccgtcgcg
     3661 caggctctcg atgagctgat gctttgggcc gaggactgcc ccgaagtccg gcacctcgtg
     3721 cacgcggatt tcggctccaa caatgtcctg acggacaatg gccgcataac agcggtcatt
     3781 gactggagcg aggcgatgtt cggggattcc caatacgagg tcgccaacat cttcttctgg
     3841 aggccgtggt tggcttgtat ggagcagcag acgcgctact tcgagcggag gcatccggag
     3901 cttgcaggat cgccgcggct ccgggcgtat atgctccgca ttggtcttga ccaactctat
     3961 cagagcttgg ttgacggcaa tttcgatgat gcagcttggg cgcagggtcg atgcgacgca
     4021 atcgtccgat ccggagccgg gactgtcggg cgtacacaaa tcgcccgcag aagcgcggcc
     4081 gtctggaccg atggctgtgt agaagtactc gccgatagtg gaaaccgacg ccccagcact
     4141 cgtccgaggg caaaggaata gcacgtacta cgagatttcg attccaccgc cgccttctat
     4201 gaaaggttgg gcttcggaat cgttttccgg gacgccggct ggatgatcct ccagcgcggg
     4261 gatctcatgc tggagttctt cgcccacccc aacttgttta ttgcagctta taatggttac
     4321 aaataaagca atagcatcac aaatttcaca aataaagcat ttttttcact gcattctagt
     4381 tgtggtttgt ccaaactcat caatgtatct tatcatgtct gtataccgtc gacctctagc
     4441 tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt gaaattgtta tccgctcaca
     4501 attccacaca acatacgagc cggaagcata aagtgtaaag cctggggtgc ctaatgagtg
     4561 agctaactca cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg
     4621 tgccagctgc attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc
     4681 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
     4741 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
     4801 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
     4861 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
     4921 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
     4981 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
     5041 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
     5101 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
     5161 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
     5221 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
     5281 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
     5341 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
     5401 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
     5461 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
     5521 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
     5581 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
     5641 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     5701 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     5761 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     5821 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     5881 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     5941 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     6001 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     6061 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     6121 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     6181 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     6241 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     6301 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     6361 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     6421 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     6481 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     6541 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     6601 aaagtgccac ctgacgtcga cggatcggga gatctcccga tcccctatgg tgcactctca
     6661 gtacaatctg ctctgatgcc gcatagttaa gccagtatct gctccctgct tgtgtgttgg
     6721 aggtcgctga gtagtgcgcg agcaaaattt aagctacaac aaggcaaggc ttgaccgaca
     6781 at
//
