LOCUS       sequence-468485-        4874 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Repair template for inserting FLAG-TEV protease cleavage site-AviTag
            into the C terminus of mouse Rpl31 via CRISPR.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4874)
  AUTHORS   Zhang Z, Xu A, Bai Y, Chen Y, Cates K, Kerr C, Bermudez A, Susanto 
            TT, Wysong K, Garcia Marques FJ, Nolan GP, Pitteri S, Barna M
  TITLE     A subcellular map of translational machinery composition and 
            regulation at the single-molecule level.
  JOURNAL   Science. 2025 Mar 7;387(6738):eadn2623. doi: 
            10.1126/science.adn2623. Epub 2025 Mar 7.
  PUBMED    40048539
REFERENCE   2  (bases 1 to 4874)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Science."; 
            date: "2025-03-7"; volume: "387(6738):eadn2623. doi"; pages: " 
            10.1126/science.adn2623. Epub 2025 Mar 7"
COMMENT     Sequence Label: pCR4-TOPO_L31-FLAG-TEV-AviTag_repair_template
FEATURES             Location/Qualifiers
     source          1..4874
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    107..128
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(143..148,149..166,167..173)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    181..197
                     /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)."
     primer_bind     186..208
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     205..221
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     205..221
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        242..260
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             complement(709..753)
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     CDS             complement(769..789)
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             complement(799..822)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     primer_bind     complement(1246..1265)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(1247..1265)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(1272..1289)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1272..1288)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1281..1303)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             1426..1728
                     /codon_start=1
                     /gene="ccdB"
                     /product="CcdB, a bacterial toxin that poisons DNA gyrase"
                     /label=ccdB
                     /note="Plasmids containing the ccdB gene cannot be 
                     propagated in standard E. coli strains."
                     /translation="QFKVYTYKRESRYRLFVDVQSDIIDTPGRRMVIPLASARLLSDKV
                     SRELYPVVHIGDESWRMMTTDMASVPVSVIGEEVADLSHRENDIKNAINLMFWGI"
     primer_bind     1607..1626
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             2077..2871
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     CDS             2077..2871
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(2131..2150)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     2741..2760
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             complement(join(3121..3912,3913..3981))
                     /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     3744..3763
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4105..4693
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4594..4613
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4847..4864
                     /label=L4440
                     /note="L4440 vector, forward primer"
ORIGIN
        1 agcgcccaat acgcaaaccg cctctccccg cgcgttggcc gattcattaa tgcagctggc
       61 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaattaat gtgagttagc
      121 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa
      181 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gccaagctca
      241 gaattaaccc tcactaaagg gactagtcct gcaggtttaa acgaattcgc ccttcactga
      301 gcctccacct agcctacgtt gaccgtctgt agatctggac ctaatcttag gctcacacat
      361 acacctggaa ctgaaaatga gagcttacaa acaagctggg catttaattg ccaatactag
      421 ggaggaaaag gcaggtggat ttctgagttc aaggccaccc tagtctatac aacaagcagt
      481 gccacacaca agaaaccctc tttcaaaacc aacccaaatc ctaataaagt gacttctgtg
      541 aaaagaatgt ttcacgtatt tctaatttcc catgtaccca gaacagagat tctcagatta
      601 atttccaaca ccctgtagac ccaaaaattc cacccatgac gcagacaaca gaaaggaaaa
      661 ccaaaccctg aaggcagttc tgcaacttta tttgacacga gcaggttact cgtgccattc
      721 aatcttctgg gcctcgaaga tgtcgttcag gcctccactg ccagaaccgc cctggaagta
      781 caggttctca ccagatccct tgtcgtcatc gtctttgtaa tcgccgctag atccactacc
      841 gttctcatcc acgttgaccg tctgaagatc tggaaggaga gtaacagcca gtcagcaccc
      901 acgcatcctg ccccagctgg aggccactgc acaacccatg gcctgtgccg cccttcccag
      961 caacctctgt actatcttcc cccagccagt tagtttttaa ggaacatctg ataatttaac
     1021 ctgctgctta cctcttcact gaacatagtt aagacacaca tcatctcaca tacaataact
     1081 cctgcggaac gctgggatac ttacttttga atgtggtaac aggcacgtaa gttaccagcg
     1141 tgtagagctt gtttggggaa tcctcatcct catgggtgga atttttgggt aggtcaggcg
     1201 gaatggcact tcaagggcga attcgcggcc gctaaattca attcgcccta tagtgagtcg
     1261 tattacaatt cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc
     1321 caacttaatc gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc
     1381 cgcaccgatc gcccttccca acagttgcgc agcctatacg tacggcagtt taaggtttac
     1441 acctataaaa gagagagccg ttatcgtctg tttgtggatg tacagagtga tattattgac
     1501 acgccggggc gacggatggt gatccccctg gccagtgcac gtctgctgtc agataaagtc
     1561 tcccgtgaac tttacccggt ggtgcatatc ggggatgaaa gctggcgcat gatgaccacc
     1621 gatatggcca gtgtgccggt ctccgttatc ggggaagaag tggctgatct cagccaccgc
     1681 gaaaatgaca tcaaaaacgc cattaacctg atgttctggg gaatataaat gtcaggcatg
     1741 agattatcaa aaaggatctt cacctagatc cttttcacgt agaaagccag tccgcagaaa
     1801 cggtgctgac cccggatgaa tgtcagctac tgggctatct ggacaaggga aaacgcaagc
     1861 gcaaagagaa agcaggtagc ttgcagtggg cttacatggc gatagctaga ctgggcggtt
     1921 ttatggacag caagcgaacc ggaattgcca gctggggcgc cctctggtaa ggttgggaag
     1981 ccctgcaaag taaactggat ggctttcttg ccgccaagga tctgatggcg caggggatca
     2041 agctctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     2101 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     2161 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     2221 gtcaagaccg acctgtccgg tgccctgaat gaactgcaag acgaggcagc gcggctatcg
     2281 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     2341 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     2401 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     2461 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     2521 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     2581 gaactgttcg ccaggctcaa ggcgagcatg cccgacggcg aggatctcgt cgtgacccat
     2641 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     2701 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     2761 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     2821 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg aattattaac
     2881 gcttacaatt tcctgatgcg gtattttctc cttacgcatc tgtgcggtat ttcacaccgc
     2941 atcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     3001 acattcaaat atgtatccgc tcatgagatt atcaaaaagg atcttcacct agatcctttt
     3061 aaattaaaaa tgaagtttta aatcaatcta aagtatatat gagtaaactt ggtctgacag
     3121 ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc gttcatccat
     3181 agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac catctggccc
     3241 cagtgctgca atgataccgc gagacccacg ctcaccggct ccagatttat cagcaataaa
     3301 ccagccagcc ggaagggccg agcgcagaag tggtcctgca actttatccg cctccatcca
     3361 gtctattaat tgttgccggg aagctagagt aagtagttcg ccagttaata gtttgcgcaa
     3421 cgttgttgcc attgctacag gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt
     3481 cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc
     3541 ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag tgttatcact
     3601 catggttatg gcagcactgc ataattctct tactgtcatg ccatccgtaa gatgcttttc
     3661 tgtgactggt gagtactcaa ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg
     3721 ctcttgcccg gcgtcaatac gggataatac cgcgccacat agcagaactt taaaagtgct
     3781 catcattgga aaacgttctt cggggcgaaa actctcaagg atcttaccgc tgttgagatc
     3841 cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta ctttcaccag
     3901 cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa taagggcgac
     3961 acggaaatgt tgaatactca tactcttcct ttttcaatat tattgaagca tttatcaggg
     4021 ttattgtctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     4081 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     4141 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     4201 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     4261 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     4321 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     4381 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     4441 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     4501 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     4561 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     4621 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     4681 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     4741 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4801 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4861 cgaggaagcg gaag
//
