LOCUS       sequence-432482-        8155 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Insect cell expression vector for FLAG-TEV-Spy-hsCRBN.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8155)
  AUTHORS   Mercer JAM, DeCarlo SJ, Roy Burman SS, Sreekanth V, Nelson AT, 
            Hunkeler M, Chen PJ, Donovan KA, Kokkonda P, Tiwari PK, Shoba VM, 
            Deb A, Choudhary A, Fischer ES, Liu DR
  TITLE     Continuous evolution of compact protein degradation tags regulated 
            by selective molecular glues.
  JOURNAL   Science. 2024 Mar 15;383(6688):eadk4422. doi: 
            10.1126/science.adk4422. Epub 2024 Mar 15.
  PUBMED    38484051
REFERENCE   2  (bases 1 to 8155)
  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: "Science."; 
            date: "2024-03-15"; volume: "383(6688):eadk4422. doi"; pages: " 
            10.1126/science.adk4422. Epub 2024 Mar 15"
COMMENT     Sequence Label: pSSRB070_pAC8-hsCRBN
FEATURES             Location/Qualifiers
     source          1..8155
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        35..126
                     /gene="polh from Autographa californica"
                     /label=polyhedrin promoter
                     /note="promoter for the baculovirus polyhedrin gene"
     primer_bind     51..68
                     /label=Polyhedrin-F
                     /note="Polyhedrin promoter, forward primer"
     CDS             141..164
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             177..197
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             216..254
                     /codon_start=1
                     /product="peptide derived from the CnaB2 adhesin domain of 
                     Streptococcus pyogenes FbaB"
                     /label=SpyTag
                     /note="forms a covalent isopeptide bond with the SpyCatcher
                     protein (Zakeri et al., 2012)"
                     /translation="AHIVMVDAYKPTK"
     misc_recomb     1632..2989
                     /label=baculovirus recombination region (ORF1629)
                     /note="contains part of ORF1629"
     CDS             complement(1648..2989)
                     /codon_start=2
                     /product="baculovirus capsid-associated protein"
                     /label=ORF1629
                     /note="required for viral replication"
                     /translation="PSLRQSLYNTIAYIERLLNIGTVNDSEITMLIADFYDLYSNYNIE
                     LPPPQALPRSRRPSVVQPAAPAPVPTIVREQTKPEQIIPAAPPPPPSPVPNIPAPPPPP
                     PPSMSELPPAPPMPTEPQPAAPLDDRQQLLEAIRNEKNRTRLRPVKPKTAPETSTIVEV
                     PTVLPKETFEPKPPSASPPPPPPPPPPPAPPAPPPMVDLSSAPPPPPLVDLPSEMLPPP
                     APSLSNVLSELKSGTVRLKPAQKRPQSEIIPKSSTTNLIADVLADTINRRRVAMAKSSS
                     EATSNDEGWDDDDNRPNKANTPDVKYVQALFNVFTSSQLYTNDSDERNTKAHNILNDVE
                     PLLQNKTQTNIDKARLLLQDLASFVALSENPLDSPAIGSEKQPLFETNRNLFYKSIEDL
                     IFKFRYKDAENHLIFALTYHPKDYKFNELLKYVQQLSVNQQRTESSA"
     rep_origin      3136..3591
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3223..3242)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3433..3454
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3608..3627)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3727..3749
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3787..3805)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3873..3977
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3978..4046,4047..4838)
                     /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     complement(4196..4215)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5009..5597
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5498..5517
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5751..5768
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_recomb     6160..6987
                     /label=baculovirus recombination region (lef2/ORF603)
                     /note="contains ORF603 and part of lef2"
     CDS             6160..6284
                     /codon_start=3
                     /product="baculovirus late expression factor 2"
                     /label=lef2
                     /translation="HLLIKSQDVYKPPNCQKMKTVDKLCPFAGNCKGLNPICNY"
     CDS             complement(6322..6927)
                     /codon_start=1
                     /product="baculovirus ORF603 protein"
                     /label=ORF603
                     /translation="MAVIFNNKQLLADNSIEKGGELFLFNGSYNILESYVNPVLLKNGV
                     IELEEAAYYAGNILYKTDDPKFIDYINLIIKATHSEELPENSTVVNYRKTMRSGTIHPI
                     KKDIYIYDNKKFTLYDRYIYGYDNNYVNFYEEKNEKEKEYEEEDDKASSLCENKIILSQ
                     INCESFENDFKYYLSDYNYAFSIIDNTTNVLVAFGLYR"
     protein_bind    complement(6991..7024)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     polyA_signal    7214..7262
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     primer_bind     7287..7306
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     CDS             complement(7340..8017)
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Strongin et al., 2007)"
                     /label=DsRed-Monomer
                     /note="mammalian codon-optimized"
                     /translation="MDNTEDVIKEFMQFKVRMEGSVNGHYFEIEGEGEGKPYEGTQTAK
                     LQVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYMKLSFPEGFTWERSMNFEDGGV
                     VEVQQDSSLQDGTFIYKVKFKGVNFPADGPVMQKKTAGWEPSTEKLYPQDGVLKGEISH
                     ALKLKDGGHYTCDFKTVYKAKKPVQLPGNHYVDSKLDITNHNEDYTVVEQYEHAEARHS
                     GSQ"
     primer_bind     complement(7600..7619)
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     promoter        complement(8026..8135)
                     /label=p10 promoter
                     /note="baculovirus promoter for expression in insect cells"
ORIGIN
        1 cgacctactc cggaatatta atagagttgc tgatatcatg gagataatta aaatgataac
       61 catctcgcaa ataaataagt attttactgt tttcgtaaca gttttgtaat aaaaaaacct
      121 ataaatacgg atctaccatg gactacaaag acgatgacga caagtccgcg gtcgacgaga
      181 acctctactt ccagggaggc ggccgcggag gctctgctca tatcgtgatg gtagacgcat
      241 ataaaccaac caaaggtgga agtggaatgg ctggtgaggg cgaccaacaa gatgcagctc
      301 acaatatggg aaatcatctc ccactgttgc cagccgaatc tgaagaagag gacgagatgg
      361 aagtcgagga tcaagactcc aaggaagcta agaagccgaa tattatcaac ttcgatacat
      421 cgctccctac atcccacact tatttgggcg ccgacatgga agagttccac ggtcgcaccc
      481 ttcatgacga tgatagttgc caagtaatcc ccgtactccc tcaggtcatg atgattctta
      541 tacctggtca aaccctgccg ctgcaactat tccatccgca agaggtgtcc atggtccgaa
      601 acctgatcca aaaggaccgt acgttcgcag tgttggccta ttcaaacgtc caagagcgtg
      661 aggcccaatt cggcaccact gctgaaatct acgcgtaccg tgaggagcaa gacttcggca
      721 tcgaaattgt aaaggtcaag gcgatcggcc gtcagcgctt taaggtgctg gaattgcgca
      781 ctcaatccga cggtatacaa caggcaaagg ttcagatctt gccagagtgc gtcctgccgt
      841 cgactatgtc ggcggtacag ctggagagtc tgaacaaatg tcaaatcttc ccctcgaagc
      901 cagtgtctcg cgaggatcag tgcagctaca agtggtggca aaagtatcag aaaaggaaat
      961 tccactgcgc taacttgacc tcgtggccga ggtggctcta cagcctttac gacgcggaaa
     1021 ctctgatgga tcgtataaag aagcaactca gggagtggga cgagaacttg aaggacgaca
     1081 gcctacccag taaccctatt gacttctcct atcgtgtcgc agcctgcctg ccgatcgacg
     1141 acgtgctgag gatacaattg ctgaagatcg gatcagcgat tcaacgcttg cgttgcgagc
     1201 tcgatatcat gaacaagtgc acctctctct gttgcaagca gtgccaggag accgagatta
     1261 ccactaagaa cgagatcttt tcattgtcac tctgcggacc aatggctgcg tacgtaaacc
     1321 cccacggcta cgtacacgaa accctcacag tttacaaagc ctgtaatctc aacctaatcg
     1381 gtcgcccaag cactgagcat tcttggttcc cgggttacgc ttggaccgtg gctcaatgca
     1441 aaatttgcgc tagtcacatc ggttggaaat tcaccgctac aaagaaggat atgtcgccgc
     1501 agaagttctg gggtctaact aggagcgcac tccttccaac catacctgat acggaagacg
     1561 agatttcgcc tgataaggtg attctgtgtc tgtaaggtac ccccggacgg ccgcttaatt
     1621 aattgatccg ggttattagt acatttatta agcgctagat tctgtgcgtt gttgatttac
     1681 agacaattgt tgtacgtatt ttaataattc attaaattta taatctttag ggtggtatgt
     1741 tagagcgaaa atcaaatgat tttcagcgtc tttatatctg aatttaaata ttaaatcctc
     1801 aatagatttg taaaataggt ttcgattagt ttcaaacaag ggttgttttt ccgaaccgat
     1861 ggctggacta tctaatggat tttcgctcaa cgccacaaaa cttgccaaat cttgtagcag
     1921 caatctagct ttgtcgatat tcgtttgtgt tttgttttgt aataaaggtt cgacgtcgtt
     1981 caaaatatta tgcgcttttg tatttctttc atcactgtcg ttagtgtaca attgactcga
     2041 cgtaaacacg ttaaataaag cttggacata tttaacatcg ggcgtgttag ctttattagg
     2101 ccgattatcg tcgtcgtccc aaccctcgtc gttagaagtt gcttccgaag acgattttgc
     2161 catagccaca cgacgcctat taattgtgtc ggctaacacg tccgcgatca aatttgtagt
     2221 tgagcttttt ggaattattt ctgattgcgg gcgtttttgg gcgggtttca atctaactgt
     2281 gcccgatttt aattcagaca acacgttaga aagcgatggt gcaggcggtg gtaacatttc
     2341 agacggcaaa tctactaatg gcggcggtgg tggagctgat gataaatcta ccatcggtgg
     2401 aggcgcaggc ggggctggcg gcggaggcgg aggcggaggt ggtggcggtg atgcagacgg
     2461 cggtttaggc tcaaatgtct ctttaggcaa cacagtcggc acctcaacta ttgtactggt
     2521 ttcgggcgcc gtttttggtt tgaccggtct gagacgagtg cgattttttt cgtttctaat
     2581 agcttccaac aattgttgtc tgtcgtctaa aggtgcagcg ggttgaggtt ccgtcggcat
     2641 tggtggagcg ggcggcaatt cagacatcga tggtggtggt ggtggtggag gcgctggaat
     2701 gttaggcacg ggagaaggtg gtggcggcgg tgccgccggt ataatttgtt ctggtttagt
     2761 ttgttcgcgc acgattgtgg gcaccggcgc aggcgccgct ggctgcacaa cggaaggtcg
     2821 tctgcttcga ggcagcgctt ggggtggtgg caattcaata ttataattgg aatacaaatc
     2881 gtaaaaatct gctataagca ttgtaatttc gctatcgttt accgtgccga tatttaacaa
     2941 ccgctcaatg taagcaattg tattgtaaag agattgtctc aagctcggat cgatcccgca
     3001 cgccgataac aagccttttc atttttacta cagcattgta gtggcgagac acttcgctgt
     3061 cgtcgcctga tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt
     3121 caaagcaacc atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta
     3181 cgcgcagcgt gaccgctaca cttgccagcg ccctagcgcc cgctcctttc gctttcttcc
     3241 cttcctttct cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt
     3301 tagggttccg atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg
     3361 gttcacgtag tgggccatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca
     3421 cgttctttaa tagtggactc ttgttccaaa ctggaacaac actcaaccct atctcgggct
     3481 attcttttga tttataaggg attttgccga tttcggccta ttggttaaaa aatgagctga
     3541 tttaacaaaa atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca
     3601 ctctcagtac aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac
     3661 ccgctgacgc gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga
     3721 ccgtctccgg gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac
     3781 gaaagggcct cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt
     3841 agacgtcagg tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct
     3901 aaatacattc aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat
     3961 attgaaaaag gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg
     4021 cggcattttg ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg
     4081 aagatcagtt gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc
     4141 ttgagagttt tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat
     4201 gtggcgcggt attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact
     4261 attctcagaa tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca
     4321 tgacagtaag agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact
     4381 tacttctgac aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg
     4441 atcatgtaac tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg
     4501 agcgtgacac cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg
     4561 aactacttac tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg
     4621 caggaccact tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag
     4681 ccggtgagcg tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc
     4741 gtatcgtagt tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga
     4801 tcgctgagat aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat
     4861 atatacttta gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc
     4921 tttttgataa tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag
     4981 accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct
     5041 gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac
     5101 caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat actgttcttc
     5161 tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg
     5221 ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt
     5281 tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt
     5341 gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc
     5401 tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca
     5461 gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata
     5521 gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg
     5581 ggcggagcct atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct
     5641 ggccttttgc tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta
     5701 ccgcctttga gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag
     5761 tgagcgagga agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga
     5821 ttcattaatg cagaacggct ccgcccacta ttaatgaaat taaaaattcc aattttaaaa
     5881 aacgcagcaa gagaaacatt tgtatgaaag aatgcgtaga aggaaagaaa aatgtcgtcg
     5941 acatgctgaa caacaagatt aatatgcctc cgtgtataaa aaaaatattg aacgatttga
     6001 aagaaaacaa tgtaccgcgc ggcggtatgt acaggaagag gtttatacta aactgttaca
     6061 ttgcaaacgt ggtttcgtgt gccaagtgtg aaaaccgatg tttaatcaag gctctgacgc
     6121 atttctacaa ccacgactcc aagtgtgtgg gtgaagtcat gcatctttta atcaaatccc
     6181 aagatgtgta taaaccacca aactgccaaa aaatgaaaac tgtcgacaag ctctgtccgt
     6241 ttgctggcaa ctgcaagggt ctcaatccta tttgtaatta ttgaataata aaacaattat
     6301 aaatgtcaaa tttgtttttt attaacgata caaaccaaac gcaacaagaa catttgtagt
     6361 attatctata attgaaaacg cgtagttata atcgctgagg taatatttaa aatcattttc
     6421 aaatgattca cagttaattt gcgacaatat aattttattt tcacataaac tagacgcctt
     6481 gtcgtcttct tcttcgtatt ccttctcttt ttcatttttc tcctcataaa aattaacata
     6541 gttattatcg tatccatata tgtatctatc gtatagagta aattttttgt tgtcataaat
     6601 atatatgtct tttttaatgg ggtgtatagt accgctgcgc atagtttttc tgtaatttac
     6661 aacagtgcta ttttctggta gttcttcgga gtgtgttgct ttaattatta aatttatata
     6721 atcaatgaat ttgggatcgt cggttttgta caatatgttg ccggcatagt acgcagcttc
     6781 ttctagttca attacaccat tttttagcag caccggatta acataacttt ccaaaatgtt
     6841 gtacgaaccg ttaaacaaaa acagttcacc tcccttttct atactattgt ctgcgagcag
     6901 ttgtttgttg ttaaaaataa cagccattgt aatgagacgc acaaactaat atcacaaact
     6961 ggaaatgtct atcaatatat agttgctgat ataacttcgt atagcataca ttatacgaag
     7021 ttatctggtt gaaacgtacc cgtagtggct atggcagggc ttgccgcccc gacgttggct
     7081 gcgagccctg ggccttcacc cgaacttggg ggttggggtg gggaaaagga agaaacgcgg
     7141 gcgtattggt cccaatgggg tctcggtggg gtatcgacag agtgccagcc ctgggaccga
     7201 accccgcgtt tatgaacaaa cgacccaaca cccgtgcgtt ttattctgtc tttttattgc
     7261 cgtcatagcg cgggttcctt ccggtattgt ctccttccgt gtttcagtta gcctccccca
     7321 tctcccgcta ttactcgagt cactgggagc cggagtggcg ggcctcggcg tgctcgtact
     7381 gctccaccac ggtgtagtcc tcgttgtggt tggtgatgtc cagcttggag tccacgtagt
     7441 ggttgccggg cagctgcacg ggcttcttgg ccttgtacac ggtcttgaag tcgcaggtgt
     7501 agtggccgcc gtccttcagc ttcagggcgt gggagatctc gcccttcagc acgccgtcct
     7561 gggggtacag cttctcggtg gagggctccc agccggcagt cttcttctgc attacggggc
     7621 cgtcggcggg gaagttcacg cccttgaact tcaccttgta gatgaaggtg ccgtcctgca
     7681 gggaggagtc ctgctgcacc tccaccacgc cgccgtcctc gaagttcatg gagcgctccc
     7741 aggtgaagcc ctcggggaag gacagcttca tgtagtcggg gatgtcggcg gggtgcttca
     7801 cgtaggcctt ggagccgtac tggaactggg gggacaggat gtcccaggcg aagggcaggg
     7861 ggccgccctt ggtcacctgc agcttggcgg tctgggtgcc ctcgtagggc ttgccctcgc
     7921 cctcgccctc gatctcgaag tagtggccgt tcacggagcc ctccatgcgc accttgaact
     7981 gcatgaactc cttgatgacg tcctcggtgt tgtccatggt agagtgattg taaataaaat
     8041 gtaatttaca gtatagtatt ttaattaata tacaaatgat ttgataataa ttcttattta
     8101 actataatat attgtgttgg gttgaattaa aggtccgtat actagtatcg attcg
//
