LOCUS       sequence-230643-        6713 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 6713)
  TITLE     Otonkoski plasmids 2018
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6713)
  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            "Unpublished"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..6713
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(12..29)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(183..771)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(263..282)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(942..1733,1734..1802))
                     /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     1565..1584
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(1803..1907)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     1975..1993
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(2031..2053)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     2153..2172
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2366..2388
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     2380..2397
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     2381..2397
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             2723..2776
                     /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             2786..2806
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             2816..2836
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             join(2843..2845,2846..2848,2849..3559)
                     /codon_start=1
                     /product="Emerald GFP"
                     /label=EmGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTFTYGVQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHKVYITADKQKNGIK
                     VNFKTRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2888..2909)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3149..3168)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3496..3517
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             3569..3625
                     /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             3626..4219
                     /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(4256..4272)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    4280..4296
                     /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    4349..4382
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     promoter        4443..4939
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(4478..4501)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     4872..4891
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     CDS             4979..5578
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     protein_bind    6135..6168
                     /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(6494..6510)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     protein_bind    6518..6534
                     /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(6542..6548,6549..6566,6567..6572))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6587..6608
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
ORIGIN
        1 tcttccgctt cctcgctcac tgactcgctg cgctcggtcg ttcggctgcg gcgagcggta
       61 tcagctcact caaaggcggt aatacggtta tccacagaat caggggataa cgcaggaaag
      121 aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta aaaaggccgc gttgctggcg
      181 tttttccata ggctccgccc ccctgacgag catcacaaaa atcgacgctc aagtcagagg
      241 tggcgaaacc cgacaggact ataaagatac caggcgtttc cccctggaag ctccctcgtg
      301 cgctctcctg ttccgaccct gccgcttacc ggatacctgt ccgcctttct cccttcggga
      361 agcgtggcgc tttctcatag ctcacgctgt aggtatctca gttcggtgta ggtcgttcgc
      421 tccaagctgg gctgtgtgca cgaacccccc gttcagcccg accgctgcgc cttatccggt
      481 aactatcgtc ttgagtccaa cccggtaaga cacgacttat cgccactggc agcagccact
      541 ggtaacagga ttagcagagc gaggtatgta ggcggtgcta cagagttctt gaagtggtgg
      601 cctaactacg gctacactag aagaacagta tttggtatct gcgctctgct gaagccagtt
      661 accttcggaa aaagagttgg tagctcttga tccggcaaac aaaccaccgc tggtagcggt
      721 ggtttttttg tttgcaagca gcagattacg cgcagaaaaa aaggatctca agaagatcct
      781 ttgatctttt ctacggggtc tgacgctcag tggaacgaaa actcacgtta agggattttg
      841 gtcatgagat tatcaaaaag gatcttcacc tagatccttt taaattaaaa atgaagtttt
      901 aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg cttaatcagt
      961 gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg actccccgtc
     1021 gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc aatgataccg
     1081 cgagacccac gctcaccggc tccagattta tcagcaataa accagccagc cggaagggcc
     1141 gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa ttgttgccgg
     1201 gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc cattgctaca
     1261 ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg ttcccaacga
     1321 tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc cttcggtcct
     1381 ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat ggcagcactg
     1441 cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg tgagtactca
     1501 accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc ggcgtcaata
     1561 cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg aaaacgttct
     1621 tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat gtaacccact
     1681 cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg gtgagcaaaa
     1741 acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg ttgaatactc
     1801 atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct catgagcgga
     1861 tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac atttccccga
     1921 aaagtgccac ctgacgtcta agaaaccatt attatcatga cattaaccta taaaaatagg
     1981 cgtatcacga ggccctttcg tctcgcgcgt ttcggtgatg acggtgaaaa cctctgacac
     2041 atgcagctcc cggagacggt cacagcttgt ctgtaagcgg atgccgggag cagacaagcc
     2101 cgtcagggcg cgtcagcggg tgttggcggg tgtcggggct ggcttaacta tgcggcatca
     2161 gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag atgcgtaagg
     2221 agaaaatacc gcatcaggcg ccattcgcca ttcaggctgc gcaactgttg ggaagggcga
     2281 tcggtgcggg cctcttcgct attacgccag ctggcgaaag ggggatgtgc tgcaaggcga
     2341 ttaagttggg taacgccagg gttttcccag tcacgacgtt gtaaaacgac ggccagtgaa
     2401 ttgctcgagc aggtatttca cagtacaccc tttaaaaaca gaatccttta ctgtttcctc
     2461 aagacacttg tgcatgttac cagtggtaga caatctgtga tcatttattg aaaatatcta
     2521 atcaaacgct aattttaaca cttattttct tagtttccca gaagaggtcg ccattgttga
     2581 agaacttggc cgcagacatg ctgatggttc tttctctgat gagatgaaca ccattcttga
     2641 taatcttgca gccagggact ttataaactg gttgattcag accaaaatca ctgacaggaa
     2701 agctagcgtc gacggctccg gagagggcag aggaagtctg ctaacatgcg gtgacgtcga
     2761 ggagaatcct ggcccaatcg atggtccaaa aaagaagaga aaggtattcg atggtccaaa
     2821 aaagaagaga aaggtattcg atatggtgag caagggcgag gagctgttca ccggggtggt
     2881 gcccatcctg gtcgagctgg acggcgacgt aaacggccac aagttcagcg tgtccggcga
     2941 gggcgagggc gatgccacct acggcaagct gaccctgaag ttcatctgca ccaccggcaa
     3001 gctgcccgtg ccctggccca ccctcgtgac caccttcacc tacggcgtgc agtgcttcgc
     3061 ccgctacccc gaccacatga agcagcacga cttcttcaag tccgccatgc ccgaaggcta
     3121 cgtccaggag cgcaccatct tcttcaagga cgacggcaac tacaagaccc gcgccgaggt
     3181 gaagttcgag ggcgacaccc tggtgaaccg catcgagctg aagggcatcg acttcaagga
     3241 ggacggcaac atcctggggc acaagctgga gtacaactac aacagccaca aggtctatat
     3301 caccgccgac aagcagaaga acggcatcaa ggtgaacttc aagacccgcc acaacatcga
     3361 ggacggcagc gtgcagctcg ccgaccacta ccagcagaac acccccatcg gcgacggccc
     3421 cgtgctgctg cccgacaacc actacctgag cacccagtcc gccctgagca aagaccccaa
     3481 cgagaagcgc gatcacatgg tcctgctgga gttcgtgacc gccgccggga tcactctcgg
     3541 catggacgag ctgtacaagg gatccggcgc aacaaacttc tctctgctga aacaagccgg
     3601 agatgtcgaa gagaatcctg gaccgaccga gtacaagccc acggtgcgcc tcgccacccg
     3661 cgacgacgtc cccagggccg tacgcaccct cgccgccgcg ttcgccgact accccgccac
     3721 gcgccacacc gtcgatccgg accgccacat cgagcgggtc accgagctgc aagaactctt
     3781 cctcacgcgc gtcgggctcg acatcggcaa ggtgtgggtc gcggacgacg gcgccgcggt
     3841 ggcggtctgg accacgccgg agagcgtcga agcgggggcg gtgttcgccg agatcggccc
     3901 gcgcatggcc gagttgagcg gttcccggct ggccgcgcag caacagatgg aaggcctcct
     3961 ggcgccgcac cggcccaagg agcccgcgtg gttcctggcc accgtcggag tctcgcccga
     4021 ccaccagggc aagggtctgg gcagcgccgt cgtgctcccc ggagtggagg cggccgagcg
     4081 cgccggggtg cccgccttcc tggagacctc cgcgccccgc aacctcccct tctacgagcg
     4141 gctcggcttc accgtcaccg ccgacgtcga ggtgcccgaa ggaccgcgca cctggtgcat
     4201 gacccgcaag cccggtgccg tcgactagat gcagcccaag cttggcgtaa tcatggtcat
     4261 agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc acacaacata cgagccggca
     4321 attccgatca tattcaataa cccttaatat aacttcgtat aatgtatgct atacgaagtt
     4381 attaggtctg aagaggagtt tacgtccagc caagcttagg atctcgacct cgaaattcta
     4441 ccgggtaggg gaggcgcttt tcccaaggca gtctggagca tgcgctttag cagccccgct
     4501 ggcacttggc gctacacaag tggcctctgg cctcgcacac attccacatc caccggtagc
     4561 gccaaccggc tccgttcttt ggtggcccct tcgcgccacc ttctactcct cccctagtca
     4621 ggaagttccc ccccgccccg cagctcgcgt cgtgcaggac gtgacaaatg gaagtagcac
     4681 gtctcactag tctcgtgcag atggacagca ccgctgagca atggaagcgg gtaggccttt
     4741 ggggcagcgg ccaatagcag ctttgctcct tcgctttctg ggctcagcag ctgggaaggg
     4801 tgggtccggg ggcgggctca ggggcgggct caggggcggg gcgggcgccc gaaggtcctc
     4861 cggaggcccg gcattctgca cgcttcaaaa gcgcacgtct gccgcgctgt tctcctcttc
     4921 ctcatctccg ggcctttcga cctgcatcca tctagatctc gagcagctga agcttaccat
     4981 gaccgagtac aagcccacgg tgcgcctcgc cacccgcgac gacgtcccca gggccgtacg
     5041 caccctcgcc gccgcgttcg ccgactaccc cgccacgcgc cacaccgtcg atccggaccg
     5101 ccacatcgag cgggtcaccg agctgcaaga actcttcctc acgcgcgtcg ggctcgacat
     5161 cggcaaggtg tgggtcgcgg acgacggcgc cgcggtggcg gtctggacca cgccggagag
     5221 cgtcgaagcg ggggcggtgt tcgccgagat cggcccgcgc atggccgagt tgagcggttc
     5281 ccggctggcc gcgcagcaac agatggaagg cctcctggcg ccgcaccggc ccaaggagcc
     5341 cgcgtggttc ctggccaccg tcggcgtctc gcccgaccac cagggcaagg gtctgggcag
     5401 cgccgtcgtg ctccccggag tggaggcggc cgagcgcgcc ggggtgcccg ccttcctgga
     5461 gacctccgcg ccccgcaacc tccccttcta cgagcggctc ggcttcaccg tcaccgccga
     5521 cgtcgaggtg cccgaaggac cgcgcacctg gtgcatgacc cgcaagcccg gtgcctgacg
     5581 cccgccccac gacccgcagc gcccgaccga aaggagcgca cgaccccatg catcgatgat
     5641 atcagatccc cgggatgcag aaattgatga tctattaaac aataaagatg tccactaaaa
     5701 tggaagtttt tcctgtcata ctttgttaag aagggtgaga acagagtacc tacattttga
     5761 atggaaggat tggagctacg ggggtggggg tggggtggga ttagataaat gcctgctctt
     5821 tactgaaggc tctttactat tgctttatga taatgtttca tagttggata tcataattta
     5881 aacaagcaaa accaaattaa gggccagctc attcctccca ctcatgatct atagatctat
     5941 agatctctcg tgggatcatt gtttttctct tgattcccac tttgtggttc taagtactgt
     6001 ggtttccaaa tgtgtcagtt tcatagcctg aagaacgaga tcagcagcct ctgttccaca
     6061 tacacttcat tctcagtatt gttttgccaa gttctaattc catcagaagc tggtcgagat
     6121 ccggaaccct taatataact tcgtataatg tatgctatac gaagttatta ggtccctcga
     6181 agaggttcac taggcgcgcc gactttataa actggttgat tcagaccaaa atcactgaca
     6241 ggtgactgct ttttagttaa ttctgaaaac catcaaattc tcataagtac tgattgtcat
     6301 actgctgcaa gctgttcccc atgtaggggg aaagtctata atctcttatt atataggaaa
     6361 taattatgca tgtttaagtc atataagagt acatcattat gtttgttttg tatcattaca
     6421 gtttgtatct atttattcat ttattccaga aacatttact gagtgtctat tagagtctag
     6481 aggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac
     6541 acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac
     6601 tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc
     6661 tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgc
//
