LOCUS       sequence-173974-        5448 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Homology arms and linker-EGFP sequence for C-terminus tagging of 
            human PXN.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5448)
  AUTHORS   Roberts B, Haupt A, Tucker A, Grancharova T, Arakaki J, Fuqua MA, 
            Nelson A, Hookway C, Ludmann SA, Mueller IA, Yang R, Horwitz R, 
            Rafelski SM, Gunawardane RN
  TITLE     Systematic gene tagging using CRISPR/Cas9 in human stem cells to 
            illuminate cell organization.
  JOURNAL   Mol Biol Cell. 2017 Oct 15;28(21):2854-2874. doi: 
            10.1091/mbc.E17-03-0209. Epub 2017 Aug 16.
  PUBMED    28814507
REFERENCE   2  (bases 1 to 5448)
  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: 
            "10.1091/mbc.E17-03-0209"; journalName: "Mol Biol Cell"; date: 
            "2017-10-15- 15"; volume: "28"; issue: "21"; pages: "2854-2874"
COMMENT     Sequence Label: AICSDP-1:PXN-EGFP
FEATURES             Location/Qualifiers
     source          1..5448
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             join(1439..1441,1442..2155)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1439..1441,1442..2152)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1481..1502)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1742..1761)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2089..2110
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(3227..3243)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(3227..3243)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(3240..3262)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    3251..3267
                     /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(3275..3281,3282..3299,3300..3305))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3320..3341
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3458..3475)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3629..4217)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3709..3728)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(4388..5179,5180..5248))
                     /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     5011..5030
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5249..5353)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     5421..5439
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt cgaaaagtgc cacctgacgt
      421 cctctgcctg ctgagttcca gtgattctcc cgcctcagcc tcccaagtag ctgagattac
      481 aggcacacgc ccccatgcct ggctaatttt ttgtattttc agtagagacg gggtttcacc
      541 atgttggcca agctggtctt gaactcctga cctcaggtga tccgcctccc ttggcctccc
      601 aaagtgctgg gattacaagt gtgagccacc tcacccggcc cctctcagag ccttttctac
      661 ctatatgtga tgtgaatctc caatgagaat ctaggaggca gagtttgact acagaccagt
      721 gtcacacctg tgtttctggg aacactgtta cagccacctg gctaagtgct caggagtcag
      781 acctgtgtat gaatccaggc tgtgacctca gtagctgcat gaccctgggc aagttacttc
      841 acctgtgtgc ttcagttgcc tcccctgttg ggagaactaa ataatcccag ccctgtggga
      901 ggccgaggtg ggaggattgc aggaggccac atttgaccag catgggcaat atagtgagac
      961 ccccatctct acaaaaaaaa tttatttaat aaaataaaaa tgaaaaatga gcgtttagga
     1021 caacacggca catgggaaac gcctagcaag taggagtcac tccgagcgtg ccgactatgc
     1081 ccacctcggc cccatcacac agggtgcagc tctagcccga ggggcagctc cctgagcccc
     1141 tctctccgcc tggcaggaat gcttcacgcc attcgtgaac ggcagcttct tcgagcacga
     1201 cgggcagccc tactgtgagg tgcactacca cgagcggcgc ggctcgctgt gttctggctg
     1261 ccagaagccc atcaccggcc gctgcatcac cgccatggcc aagaagttcc accccgagca
     1321 cttcgtctgt gccttctgcc tcaagcagct caacaagggc aacttcaagg agcagaacga
     1381 caagccttac tgtcagaact gcttactcaa gctcttctgc ggtaccagcg gcggaagcgt
     1441 gagcaagggc gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga
     1501 cgtaaacggc cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa
     1561 gctgaccctg aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt
     1621 gaccaccctg acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca
     1681 cgacttcttc aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa
     1741 ggacgacggc aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa
     1801 ccgcatcgag ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct
     1861 ggagtacaac tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat
     1921 caaggtgaac ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca
     1981 ctaccagcag aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct
     2041 gagcacccag tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct
     2101 ggagttcgtg accgccgccg ggatcactct cggcatggac gagctgtaca agtaatgata
     2161 ggtgccctgc ccctgtctct gccccccttc cccagccagc atcaccaact gcgactgtga
     2221 cctagagact tcacccgggg gtgaaggggt aaacccgact gaaactggaa cccttgtcct
     2281 ccgctggtgc gggatggaca gagggccgtg aggggtcccc ctgcttgtct tcacccctgc
     2341 cagagcctct gggccccctc ctccctcctg tagctctccc taggctgccc actctccatc
     2401 ctccccaggg gtagaggctg ggggctccac cccagcccat gtacgtcccc acgaactggc
     2461 ctggccagca ccccacactg gagccatctc ttcctcatat ttcagcagtg cagccggggg
     2521 gcagggaagg gcaggcaggg tctgttgggg tctcttttta tccttattcc tcccccgacc
     2581 taattgtctt tgttctgtga ttattggggg acacccggct ccctccagac aatgccagca
     2641 taaatccatc catccaaagg cagagaacca aaggggccat ggaaggttct ctgtgctcct
     2701 cctacccttc cagtgcccta ggcctggcga ctgcccctgc cttttagacc cgccctcccc
     2761 ttttatacct gctcttgttc tactgagaaa agcctctcca gcaataatgt tttctagtca
     2821 cttcctccgt ctccgtgacg gcgtgcctgg acactgtacc gactttgata gatttctaca
     2881 ctgaggtttg aattcatatc gcctgagttg cttttacttc tctatacaaa atgattttga
     2941 agagatttta aagacgttcc cttttgtatt ctcttcctca tccaccgcca ctgggcctgt
     3001 cactgatggt ggctctggtg tgaagtttgc tttgtactga gggttggggt ggggaagcaa
     3061 tttgtatttt attgtttctt agcacaagca ggtgaactgg gagcagctct gtgactcccc
     3121 ctctttcact tcatagctca ccaggactgt tttataaact acgtctaaga aaccattatt
     3181 atcctctaga gtcgacctgc aggcatgcaa gcttggcgta atcatggtca tagctgtttc
     3241 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt
     3301 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc
     3361 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg
     3421 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgac tcgctgcgct
     3481 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca
     3541 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga
     3601 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc
     3661 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg
     3721 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat
     3781 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt
     3841 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc
     3901 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg
     3961 acttatcgcc actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg
     4021 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg
     4081 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg
     4141 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca
     4201 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga
     4261 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga
     4321 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt
     4381 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt
     4441 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat
     4501 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctcca gatttatcag
     4561 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct
     4621 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt
     4681 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg
     4741 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca
     4801 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt
     4861 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat
     4921 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac
     4981 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa
     5041 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt
     5101 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt
     5161 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa
     5221 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt
     5281 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa
     5341 taggggttcc gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta
     5401 tcatgacatt aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
