LOCUS       sequence-235290-        6716 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor template for CRISPR-Cas9 mediated generation of GCG-NLS-EmGFP 
            reporter cell lines.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6716)
  TITLE     Otonkoski plasmids 2018
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6716)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pUC19-GCG-T2A-NLS-EmGFP-P2A-puro-PGK-puro
FEATURES             Location/Qualifiers
     source          1..6716
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     CDS             326..379
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             389..409
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             419..439
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(446..448,449..451,452..1162)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(491..512)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(752..771)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1099..1120
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1172..1228
                     /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             1229..1822
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="TEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIERV
                     TELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLAA
                     QQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETSA
                     PRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1859..1875)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    1883..1899
                     /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)."
     protein_bind    1952..1985
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        2046..2545
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2081..2104)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2478..2497
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             2585..3184
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APHNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     protein_bind    3741..3774
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     complement(4100..4116)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4124..4140
                     /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(4148..4154,4155..4172,4173..4178))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4193..4214
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4331..4348)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4502..5090)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4582..4601)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5261..6052,6053..6121))
                     /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     5884..5903
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6122..6226)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6294..6312
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(6350..6372)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     6472..6491
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     6685..6707
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     6699..6716
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     6700..6716
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
ORIGIN
        1 gaattgctcg agcaggtatt tcacagtaca ccctttaaaa acagaatcct ttactgtttc
       61 ctcaagacac ttgtgcatgt taccagtggt agacaatctg tgatcattta ttgaaaatat
      121 ctaatcaaac gctaatttta acacttattt tcttagtttc ccagaagagg tcgccattgt
      181 tgaagaactt ggccgcagac atgctgatgg ttctttctct gatgagatga acaccattct
      241 tgataatctt gcagccaggg actttataaa ctggttgatt cagaccaaaa tcactgacag
      301 gaaagctagc gtcgacggct ccggagaggg cagaggaagt ctgctaacat gcggtgacgt
      361 cgaggagaat cctggcccaa tcgatggtcc aaaaaagaag agaaaggtat tcgatggtcc
      421 aaaaaagaag agaaaggtat tcgatatggt gagcaagggc gaggagctgt tcaccggggt
      481 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
      541 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
      601 caagctgccc gtgccctggc ccaccctcgt gaccaccttc acctacggcg tgcagtgctt
      661 cgcccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
      721 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
      781 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
      841 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaaggtcta
      901 tatcaccgcc gacaagcaga agaacggcat caaggtgaac ttcaagaccc gccacaacat
      961 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     1021 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     1081 caacgagaag cgcgatcaca tggtcctgct ggagttcgtg accgccgccg ggatcactct
     1141 cggcatggac gagctgtaca agggatccgg cgcaacaaac ttctctctgc tgaaacaagc
     1201 cggagatgtc gaagagaatc ctggaccgac cgagtacaag cccacggtgc gcctcgccac
     1261 ccgcgacgac gtccccaggg ccgtacgcac cctcgccgcc gcgttcgccg actaccccgc
     1321 cacgcgccac accgtcgatc cggaccgcca catcgagcgg gtcaccgagc tgcaagaact
     1381 cttcctcacg cgcgtcgggc tcgacatcgg caaggtgtgg gtcgcggacg acggcgccgc
     1441 ggtggcggtc tggaccacgc cggagagcgt cgaagcgggg gcggtgttcg ccgagatcgg
     1501 cccgcgcatg gccgagttga gcggttcccg gctggccgcg cagcaacaga tggaaggcct
     1561 cctggcgccg caccggccca aggagcccgc gtggttcctg gccaccgtcg gagtctcgcc
     1621 cgaccaccag ggcaagggtc tgggcagcgc cgtcgtgctc cccggagtgg aggcggccga
     1681 gcgcgccggg gtgcccgcct tcctggagac ctccgcgccc cgcaacctcc ccttctacga
     1741 gcggctcggc ttcaccgtca ccgccgacgt cgaggtgccc gaaggaccgc gcacctggtg
     1801 catgacccgc aagcccggtg ccgtcgacta gatgcagccc aagcttggcg taatcatggt
     1861 catagctgtt tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg
     1921 gcaattccga tcatattcaa taacccttaa tataacttcg tataatgtat gctatacgaa
     1981 gttattaggt ctgaagagga gtttacgtcc agccaagctt aggatctcga cctcgaaatt
     2041 ctaccgggta ggggaggcgc ttttcccaag gcagtctgga gcatgcgctt tagcagcccc
     2101 gctgggcact tggcgctaca caagtggcct ctggcctcgc acacattcca catccaccgg
     2161 taggcgccaa ccgactccgt tctttggtgg ccccttcgcg ccaccttcta ctcctcccct
     2221 agtcaggaag ttcccccccg ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt
     2281 agcacgtctc actagtctcg tgcagatgga cagcaccgct gagcaatgga agcgggtagg
     2341 cctttggggc agcggccaat agcagctttg ctccttcgct ttctgggctc agaggctggg
     2401 aaggggtggg tccgggggcg ggctcagggg cgggctcagg ggcggggcgg gcgcccgaag
     2461 gtcctccgga ggcccggcat tctgcacgct tcaaaagcgc acgtctgccg cgctgttctc
     2521 ctcttcctca tctccgggcc tttcgacctg catccatcta gatctcgagc agctgaagct
     2581 taccatgacc gagtacaagc ccacggtgcg cctcgccacc cgcgacgacg tccccagggc
     2641 cgtacgcacc ctcgccgccg cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc
     2701 ggaccgccac atcgagcggg tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct
     2761 cgacatcggc aaggtgtggg tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc
     2821 ggagagcgtc gaagcggggg cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag
     2881 cggttcccgg ctggccgcgc agcaacagat ggaaggcctc ctggcgccgc accggcccaa
     2941 ggagcccgcg tggttcctgg ccaccgtcgg cgtctcgccc gaccaccagg gcaagggtct
     3001 gggcagcgcc gtcgtgctcc ccggagtgga ggcggccgag cgcgccgggg tgcccgcctt
     3061 cctggagacc tccgcgcccc acaacctccc cttctacgag cggctcggct tcaccgtcac
     3121 cgccgacgtc gaggtgcccg aaggaccgcg cacctggtgc atgacccgca agcccggtgc
     3181 ctgacgcccg ccccacgacc cgcagcgccc gaccgaaagg agcgcacgac cccatgcatc
     3241 gatgatatca gatccccggg atgcagaaat tgatgatcta ttaaacaata aagatgtcca
     3301 ctaaaatgga agtttttcct gtcatacttt gttaagaagg gtgagaacag agtacctaca
     3361 ttttgaatgg aaggattgga gctacggggg tgggggtggg gtgggattag ataaatgcct
     3421 gctctttact gaaggctctt tactattgct ttatgataat gtttcatagt tggatatcat
     3481 aatttaaaca agcaaaacca aattaagggc cagctcattc ctcccactca tgatctatag
     3541 atctatagat ctctcgtggg atcattgttt ttctcttgat tcccactttg tggttctaag
     3601 tactgtggtt tccaaatgtg tcagtttcat agcctgaaga acgagatcag cagcctctgt
     3661 tccacataca cttcattctc agtattgttt tgccaagttc taattccatc agaagctggt
     3721 cgagatccgg aacccttaat ataacttcgt ataatgtatg ctatacgaag ttattaggtc
     3781 cctcgaagag gttcactagg cgcgccgact ttataaactg gttgattcag accaaaatca
     3841 ctgacaggtg actgcttttt agttaattct gaaaaccatc aaattctcat aagtactgat
     3901 tgtcatactg ctgcaagctg ttccccatgt agggggaaag tctataatct cttattatat
     3961 aggaaataat tatgcatgtt taagtcatat aagagtacat cattatgttt gttttgtatc
     4021 attacagttt gtatctattt attcatttat tccagaaaca tttactgagt gtctattaga
     4081 gtctagaggc gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa
     4141 ttccacacaa catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga
     4201 gctaactcac attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt
     4261 gccagctgca ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct
     4321 cttccgcttc ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat
     4381 cagctcactc aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga
     4441 acatgtgagc aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt
     4501 ttttccatag gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt
     4561 ggcgaaaccc gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc
     4621 gctctcctgt tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa
     4681 gcgtggcgct ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct
     4741 ccaagctggg ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta
     4801 actatcgtct tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg
     4861 gtaacaggat tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc
     4921 ctaactacgg ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta
     4981 ccttcggaaa aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg
     5041 gtttttttgt ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt
     5101 tgatcttttc tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg
     5161 tcatgagatt atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta
     5221 aatcaatcta aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg
     5281 aggcacctat ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg
     5341 tgtagataac tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc
     5401 gagacccacg ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg
     5461 agcgcagaag tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg
     5521 aagctagagt aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag
     5581 gcatcgtggt gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat
     5641 caaggcgagt tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc
     5701 cgatcgttgt cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc
     5761 ataattctct tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa
     5821 ccaagtcatt ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac
     5881 gggataatac cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt
     5941 cggggcgaaa actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc
     6001 gtgcacccaa ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa
     6061 caggaaggca aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca
     6121 tactcttcct ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat
     6181 acatatttga atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa
     6241 aagtgccacc tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc
     6301 gtatcacgag gccctttcgt ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca
     6361 tgcagctccc ggagacggtc acagcttgtc tgtaagcgga tgccgggagc agacaagccc
     6421 gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg gcttaactat gcggcatcag
     6481 agcagattgt actgagagtg caccatatgc ggtgtgaaat accgcacaga tgcgtaagga
     6541 gaaaataccg catcaggcgc cattcgccat tcaggctgcg caactgttgg gaagggcgat
     6601 cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg gggatgtgct gcaaggcgat
     6661 taagttgggt aacgccaggg ttttcccagt cacgacgttg taaaacgacg gccagt
//
