LOCUS       sequence-448158-        5238 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Generates GNB1L dTAG C terminal  knock-in cell lines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5238)
  AUTHORS   Huang M, Yao F, Nie L, Wang C, Su D, Zhang H, Li S, Tang M, Feng X, 
            Yu B, Chen Z, Wang S, Yin L, Mou L, Hart T, Chen J
  TITLE     FACS-based genome-wide CRISPR screens define key regulators of DNA 
            damage signaling pathways.
  JOURNAL   Mol Cell. 2023 Aug 3;83(15):2810-2828.e6. doi: 
            10.1016/j.molcel.2023.07.004.
  PUBMED    37541219
REFERENCE   2  (bases 1 to 5238)
  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: 
            "10.1016/j.molcel.2023.07"; journalName: "Mol Cell"; date: 
            "2023-08-3- 3"; volume: "83"; issue: "15"; pages: "2810-2828.e6"
COMMENT     Sequence Label: pUC19_GNB1L_dTAG
FEATURES             Location/Qualifiers
     source          1..5238
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     118..137
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     371..388
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    505..526
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(541..546,547..564,565..571)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    579..595
                     /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     584..606
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     603..619
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     603..619
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             615..938
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITPSLHACRSTLEDPRVPSSNSLAVVLQRRDWENPGVTQLNR
                     LAAHPPFASWRNSEEARTDRPSQQLRSLNGEWRLMRYFLLTHLCGISHRIWCTLSTICS
                     DAA"
     misc_feature    632..688
                     /label=MCS
                     /note="pUC18/19 multiple cloning site"
     primer_bind     complement(689..706)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(689..705)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(698..720)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(914..933)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     CDS             2018..2338
                     /codon_start=1
                     /product="F36V mutant of FK506-binding protein FKBP12"
                     /label=FKBP-V
                     /note="binds synthetic ligands that do not bind wild-type 
                     FKBP"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLEDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQRAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     KLE"
     CDS             2348..2374
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /note="This is a variant of the standard HA tag, which has 
                     sequence YPYDVPDYA. Some anti-HA antibodies may not 
                     recognize this variant."
                     /translation="YPYDVPDYA"
     CDS             2348..2374
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2378..2404
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             2408..2464
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     CDS             2465..2863
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     primer_bind     3585..3607
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3645..3663)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3731..3835
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3836..3904,3905..4696)
                     /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(4054..4073)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      join(4867..5238,1..217)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
ORIGIN
        1 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
       61 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
      121 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
      181 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
      241 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
      301 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
      361 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
      421 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
      481 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
      541 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
      601 cacaggaaac agctatgacc atgattacgc caagcttgca tgcctgcagg tcgactctag
      661 aggatccccg ggtaccgagc tcgaattcac tggccgtcgt tttacaacgt cgtgactggg
      721 aaaaccctgg cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc
      781 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg
      841 aatggcgcct gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat
      901 ggtgcactct cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc
      961 caacacccgc tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag
     1021 ctgtgaccgt ctacgtagga cctgtctgtg tggctccggg tctcgtgcag tgccctaccc
     1081 cctctcagaa cccagccggg gacgttgccc ccaccaggca aggtacactg tgtggctcct
     1141 gccgcctcca gggcgtctcc cccttgtccc cccctcatcc tgccaacccc ccactgggtc
     1201 ttgctcttgg cagcgctccc agccctgtcg agcttggggg ttcacatacc tcatggtttt
     1261 gctgaaaatg gagtttgtat ttctcaccct cgtcagcttt ggggtggttg tgaggttctt
     1321 ctgggaggag tgccagcatt ctgctgccat caagcccggg acgtggcctc tcgctaggca
     1381 ctagggtgca gtggcttccc tggggtgagg ggccccggga cccagcccct ctcctgcagc
     1441 tgggacctcc ctgtctctgc tgctctctga ctggggaagg agtcggtcag agctggtgcg
     1501 aggagcagca gggcagggcc gaagaccact ctgaggggag ggctggagtc ccccatcgtg
     1561 gccttaggaa gagtgggagg tcagctgggt aggagcgtgt ggccctcaca tcccgggcct
     1621 gtgggcctgc gtctctcctg gttccagctg caggtggggc accaggggca gcactgcctt
     1681 gtgggcttaa ggagtggcct aacacctctc ccaactctca caggtgcgtg ggactcatga
     1741 actcaccaat cccgggatcg ccgaggtcac gatccggcca gatcgcaaga tcctggccac
     1801 cgcaggctgg gaccaccgca tccgcgtgtt ccactggcgg acgatgcagc cactggccgt
     1861 gctggccttc cacagcgccg ctgtccagtg cgtggccttc accgccgatg gcttgctggc
     1921 cgcgggctcc aaggatcagc ggatcagttt atggtcactc tacccacgcg caggtggcgg
     1981 tggctcgggc ggtggtgggt cgggtggcgg cggatctgga gtgcaggtgg aaaccatctc
     2041 cccaggagac gggcgcacct tccccaagcg cggccagacc tgcgtggtgc actacaccgg
     2101 gatgcttgaa gatggaaaga aagttgattc ctcccgggac agaaacaagc cctttaagtt
     2161 tatgctaggc aagcaggagg tgatccgagg ctgggaagaa ggggttgccc agatgagtgt
     2221 gggtcagaga gccaaactga ctatatctcc agattatgcc tatggtgcca ctgggcaccc
     2281 aggcatcatc ccaccacatg ccactctcgt cttcgatgtg gagcttctaa aactggaagg
     2341 ctcgggctac ccctacgacg tgcccgacta cgccggctat ccgtatgatg tcccggacta
     2401 tgcaggcgct actaacttca gcctgctgaa gcaggctgga gacgtggagg agaaccctgg
     2461 acctatggcc aagcctttgt ctcaagaaga atccaccctc attgaaagag caacggctac
     2521 aatcaacagc atccccatct ctgaagacta cagcgtcgcc agcgcagctc tctctagcga
     2581 cggccgcatc ttcactggtg tcaatgtata tcattttact gggggacctt gtgcagaact
     2641 cgtggtgctg ggcactgctg ctgctgcggc agctggcaac ctgacttgta tcgtcgcgat
     2701 cggaaatgag aacaggggca tcttgagccc ctgcggacgg tgccgacagg tgcttctcga
     2761 tctgcatcct gggatcaaag ccatagtgaa ggacagtgat ggacagccga cggcagttgg
     2821 gattcgtgaa ttgctgccct ctggttatgt gtgggagggc tgactcaccc actcccttcc
     2881 atagagacga ggagggcggg cagggaggtg ggcatcaggc cccagccttg gcctgaaacc
     2941 tcaagggcct ctgaggacca gcaagtcatg gcccaaggac catggagtcc ccgctgtctt
     3001 catgaggttg gtatttcctt ttgtggagtt cctcatcaca ggacggtgac tctgggggcc
     3061 agccagagcc ctggccgtcc gaggcctgca ggagacgttg gcttgggcct gctgtgttgc
     3121 cagctgaggg tattttataa taaatttcca ttgccaagtt gcaggcttga ggtgggagct
     3181 ccctgaggag gccctggtcc cccacttagc atcagtgccc tcagaccccc accaggcagg
     3241 atgctcagcc ccaccctcac acctgctgtt gaatgggcca ccccgagtgt gggtttcctg
     3301 ggccaggcct caggagatga taggccccaa ccatagctgc gtcgatgtcc acatgacccc
     3361 tcaccccgag gaggccagga gctcccaaca cacaggttga ggccaggatc aggatcagga
     3421 agtaactgaa ctccctaaag agtgaagcag agaaaggtgc ctgtgaggga cggcaagggg
     3481 ggcttgcagc gcacagggcc aggccagatg tgggatccgg gagggcacgc ctgcccgcag
     3541 agggtgcagt ggggaaggcg cacagggtgc acctccaggt gagtccggga gctgcatgtg
     3601 tcagaggttt tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct
     3661 atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg
     3721 gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc
     3781 gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag
     3841 tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt
     3901 tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt
     3961 gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga
     4021 acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat
     4081 tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga
     4141 gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag
     4201 tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg
     4261 accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg
     4321 ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt
     4381 agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg
     4441 gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc
     4501 ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg ggtctcgcgg
     4561 tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac
     4621 ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact
     4681 gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa
     4741 acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa
     4801 aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg
     4861 atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc
     4921 gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac
     4981 tggcttcagc agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca
     5041 ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt
     5101 ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc
     5161 ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg
     5221 aacgacctac accgaact
//
