LOCUS       sequence-228473-        7929 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  CRISPR donor plasmid to tag human transcription factor NR1D2 with 
            GFP.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7929)
  TITLE     White lab ENCODE plasmids to tag human transcription factors with 
            GFP
REFERENCE   2  (bases 1 to 7929)
  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"
COMMENT     Sequence Label: pNR1D2-donor
FEATURES             Location/Qualifiers
     source          1..7929
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(70..89)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(71..89)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(96..113)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(96..112)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(105..127)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     CDS             250..552
                     /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     431..450
                     /label=ccdB-fwd
                     /note="ccdB gene, forward primer"
     CDS             901..1695
                     /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             901..1695
                     /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"
     regulatory      1895..1904
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1901..2275
                     /codon_start=1
                     /gene="Sh ble from Streptoalloteichus hindustanus"
                     /product="antibiotic-binding protein"
                     /label=BleoR
                     /note="confers resistance to bleomycin, phleomycin, and 
                     Zeocin(TM)"
                     /translation="MAKLTSAVPVLTARDVAGAVEFWTDRLGFSRDFVEDDFAGVVRDD
                     VTLFISAVQDQVVPDNTLAWVWVRGLDELYAEWSEVVSTNFRDASGPAMTEIGEQPWGR
                     EFALRDPAGNCVHFVAEEQD"
     rep_origin      2413..3001
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2902..2921
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3155..3172
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3289..3310
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3325..3330,3331..3348,3349..3355)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3363..3379
                     /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     3368..3390
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3387..3403
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3387..3403
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3421..3439
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3421..3438
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             4478..4498
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4511..4555
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             4595..4618
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(4646..4648,4649..4651,4652..5365)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLK
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(4691..4712)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4952..4971)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(5403..5976,5977..5979,5980..5988)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5569..5586)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5796..5815
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             6070..6873
                     /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="MGSAIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQ
                     GRPVLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQD
                     LLSSHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDE
                     EHQGLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQ
                     DIALATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
ORIGIN
        1 aagggcgaat tctgcagata tccatcacac tggcggccgc tcgagcatgc atctagaggg
       61 cccaattcgc cctatagtga gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt
      121 gactgggaaa accctggcgt tacccaactt aatcgccttg cagcacatcc ccctttcgcc
      181 agctggcgta atagcgaaga ggcccgcacc gatcgccctt cccaacagtt gcgcagccta
      241 tacgtacggc agtttaaggt ttacacctat aaaagagaga gccgttatcg tctgtttgtg
      301 gatgtacaga gtgatattat tgacacgccg gggcgacgga tggtgatccc cctggccagt
      361 gcacgtctgc tgtcagataa agtctcccgt gaactttacc cggtggtgca tatcggggat
      421 gaaagctggc gcatgatgac caccgatatg gccagtgtgc cggtctccgt tatcggggaa
      481 gaagtggctg atctcagcca ccgcgaaaat gacatcaaaa acgccattaa cctgatgttc
      541 tggggaatat aaatgtcagg catgagatta tcaaaaagga tcttcaccta gatccttttc
      601 acgtagaaag ccagtccgca gaaacggtgc tgaccccgga tgaatgtcag ctactgggct
      661 atctggacaa gggaaaacgc aagcgcaaag agaaagcagg tagcttgcag tgggcttaca
      721 tggcgatagc tagactgggc ggttttatgg acagcaagcg aaccggaatt gccagctggg
      781 gcgccctctg gtaaggttgg gaagccctgc aaagtaaact ggatggcttt cttgccgcca
      841 aggatctgat ggcgcagggg atcaagctct gatcaagaga caggatgagg atcgtttcgc
      901 atgattgaac aagatggatt gcacgcaggt tctccggccg cttgggtgga gaggctattc
      961 ggctatgact gggcacaaca gacaatcggc tgctctgatg ccgccgtgtt ccggctgtca
     1021 gcgcaggggc gcccggttct ttttgtcaag accgacctgt ccggtgccct gaatgaactg
     1081 caagacgagg cagcgcggct atcgtggctg gccacgacgg gcgttccttg cgcagctgtg
     1141 ctcgacgttg tcactgaagc gggaagggac tggctgctat tgggcgaagt gccggggcag
     1201 gatctcctgt catctcacct tgctcctgcc gagaaagtat ccatcatggc tgatgcaatg
     1261 cggcggctgc atacgcttga tccggctacc tgcccattcg accaccaagc gaaacatcgc
     1321 atcgagcgag cacgtactcg gatggaagcc ggtcttgtcg atcaggatga tctggacgaa
     1381 gagcatcagg ggctcgcgcc agccgaactg ttcgccaggc tcaaggcgag catgcccgac
     1441 ggcgaggatc tcgtcgtgac ccatggcgat gcctgcttgc cgaatatcat ggtggaaaat
     1501 ggccgctttt ctggattcat cgactgtggc cggctgggtg tggcggaccg ctatcaggac
     1561 atagcgttgg ctacccgtga tattgctgaa gagcttggcg gcgaatgggc tgaccgcttc
     1621 ctcgtgcttt acggtatcgc cgctcccgat tcgcagcgca tcgccttcta tcgccttctt
     1681 gacgagttct tctgaattat taacgcttac aatttcctga tgcggtattt tctccttacg
     1741 catctgtgcg gtatttcaca ccgcatcagg tggcactttt cggggaaatg tgcgcggaac
     1801 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     1861 ctgataaatg cttcaataat agcacgtgag gagggccacc atggccaagt tgaccagtgc
     1921 cgttccggtg ctcaccgcgc gcgacgtcgc cggagcggtc gagttctgga ccgaccggct
     1981 cgggttctcc cgggacttcg tggaggacga cttcgccggt gtggtccggg acgacgtgac
     2041 cctgttcatc agcgcggtcc aggaccaggt ggtgccggac aacaccctgg cctgggtgtg
     2101 ggtgcgcggc ctggacgagc tgtacgccga gtggtcggag gtcgtgtcca cgaacttccg
     2161 ggacgcctcc gggccggcca tgaccgagat cggcgagcag ccgtgggggc gggagttcgc
     2221 cctgcgcgac ccggccggca actgcgtgca cttcgtggcc gaggagcagg actgacacgt
     2281 gctaaaactt catttttaat ttaaaaggat ctaggtgaag atcctttttg ataatctcat
     2341 gaccaaaatc ccttaacgtg agttttcgtt ccactgagcg tcagaccccg tagaaaagat
     2401 caaaggatct tcttgagatc ctttttttct gcgcgtaatc tgctgcttgc aaacaaaaaa
     2461 accaccgcta ccagcggtgg tttgtttgcc ggatcaagag ctaccaactc tttttccgaa
     2521 ggtaactggc ttcagcagag cgcagatacc aaatactgtt cttctagtgt agccgtagtt
     2581 aggccaccac ttcaagaact ctgtagcacc gcctacatac ctcgctctgc taatcctgtt
     2641 accagtggct gctgccagtg gcgataagtc gtgtcttacc gggttggact caagacgata
     2701 gttaccggat aaggcgcagc ggtcgggctg aacggggggt tcgtgcacac agcccagctt
     2761 ggagcgaacg acctacaccg aactgagata cctacagcgt gagctatgag aaagcgccac
     2821 gcttcccgaa gggagaaagg cggacaggta tccggtaagc ggcagggtcg gaacaggaga
     2881 gcgcacgagg gagcttccag ggggaaacgc ctggtatctt tatagtcctg tcgggtttcg
     2941 ccacctctga cttgagcgtc gatttttgtg atgctcgtca ggggggcgga gcctatggaa
     3001 aaacgccagc aacgcggcct ttttacggtt cctggccttt tgctggcctt ttgctcacat
     3061 gttctttcct gcgttatccc ctgattctgt ggataaccgt attaccgcct ttgagtgagc
     3121 tgataccgct cgccgcagcc gaacgaccga gcgcagcgag tcagtgagcg aggaagcgga
     3181 agagcgccca atacgcaaac cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg
     3241 gcacgacagg tttcccgact ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta
     3301 gctcactcat taggcacccc aggctttaca ctttatgctt ccggctcgta tgttgtgtgg
     3361 aattgtgagc ggataacaat ttcacacagg aaacagctat gaccatgatt acgccaagct
     3421 atttaggtga cactatagaa tactcaagct atgcatcaag cttggtaccg agctcggatc
     3481 cactagtaac ggccgccagt gtgctggaat tcgccctttc ggcaagattg agttccttgc
     3541 aagctgttgg actgagcacc tcagttgctt gctgactgtt ggctagagcc cactcttagt
     3601 tccttgccat gtgggcctct ccacagggtg gctcacagca tggcagcagc ttttctcaga
     3661 gtaagtgagc aagagcgtac ctaagatgga ggccacagtt tcttgtaagc taatctcaga
     3721 acggacatcc tatcaattct gccatatttt ttttcattag aagttagtaa gtctaggtcc
     3781 acttagtagg aggaggttgc gcaagggtgc gaataccagg atgaagggat cattggaggc
     3841 cattttaggg gctgcctacc acacatggtg acatacaaat gggagtgtat tccatatgta
     3901 ttgaaaatat gtttatatac tacattattt tattttgcta attagaagca gttgaaatca
     3961 gatgacatct aattttaaaa agtcagaacc tctatgttct tgtgaataaa aattcttaac
     4021 agtaaagaat tttgattgat caaatgtttt atgtgatata tatgctttat aatatagtaa
     4081 ctagctaacg atttgttact gagattttga gaaatttgtc aacttttgcc tttattcaga
     4141 tgtataaaac cttgctttct attcgttagt gacactgata agcaagaatt ttatttatat
     4201 ttaatgttaa taatttatcc tgtttttcct atttccctat tcctgatctc tcttagatcg
     4261 atctggaata gaaaacgtca actctgtgga ggctttgcag gaaactctca ttcgtgcact
     4321 aaggacctta ataatgaaaa accatccaaa tgaggcctct atttttacaa aactgcttct
     4381 aaagttgcca gatcttcgat ctttaaacaa catgcactct gaggagctct tggcctttaa
     4441 agttcaccct gattatgata ttccaactac tgcaagcgag aatctttatt ttcagggcgc
     4501 cgccaaattc aaagaaaccg ctgctgctaa attcgaacgc cagcacatgg acagcggagg
     4561 tggaggttcg agcggtccct cgggttcgtc gagcctggaa gttctgttcc aggggcccct
     4621 gtcgtcgagc ggtccctcgg gttcgatggt tagcaaaggc gaggagctgt tcaccggggt
     4681 ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc cacaagttca gcgtgtccgg
     4741 cgagggcgag ggcgatgcca cctacggcaa gctgaccctg aagttcatct gcaccaccgg
     4801 caagctgccc gtgccctggc ccaccctcgt gaccaccctg acctacggcg tgcagtgctt
     4861 cagccgctac cccgaccaca tgaagcagca cgacttcttc aagtccgcca tgcccgaagg
     4921 ctacgtccag gagcgcacca tcttcttcaa ggacgacggc aactacaaga cccgcgccga
     4981 ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag ctgaagggca tcgacttcaa
     5041 ggaggacggc aacatcctgg ggcacaagct ggagtacaac tacaacagcc acaacgtcta
     5101 tatcatggcc gacaagcaga agaacggcat caaggtgaac ttcaagatcc gccacaacat
     5161 cgaggacggc agcgtgcagc tcgccgacca ctaccagcag aacaccccca tcggcgacgg
     5221 ccccgtgctg ctgcccgaca accactacct gagcacccag tccgccctga gcaaagaccc
     5281 caacgagaag cgcgatcaca tggtccttaa ggagttcgtg accgccgccg ggatcactct
     5341 cggcatggac gagctgtaca agtaatgatc tagagtcgag ttaattaaga attccgcccc
     5401 cccccctctc cctccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt
     5461 gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc
     5521 ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag
     5581 gaatgcaggg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac
     5641 aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc
     5701 tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc
     5761 acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca
     5821 aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt
     5881 gcacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg
     5941 gggacgtggt tttcctttga aaaacacgat gataatatgg ccacaacctg ttacattgca
     6001 caagataaaa atatatcatc acgaacagta aaactgtctg cttacataaa cagtaataca
     6061 aggggtgtta tgggatcggc cattgaacaa gatggattgc acgcaggttc tccggccgct
     6121 tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc
     6181 gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc
     6241 ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc
     6301 gttccttgcg cagctgtgct cgacgttgtc actgaggcgg gaagggactg gctgctattg
     6361 ggagaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga gaaagtatcc
     6421 atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac
     6481 caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat
     6541 caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc
     6601 aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg
     6661 aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg gctgggtgtg
     6721 gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga gcttggcggc
     6781 gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc gcagcgcatc
     6841 gccttctatc gccttcttga cgagttcttc tgaaggcctt tgtttattta aacatgaact
     6901 gatggtaact gtacattttg tgctaaaatg catatttata tgtgtatacc atatgtggag
     6961 atagaaaaga cctttaagac aataaaagat tgtaggctat ctctgtaatc atgcaatagc
     7021 tgttcggatt gagaactctt cagccatgat tagacgttga ctgcatctcc ctgatagacc
     7081 aatcagctgt gtcgcactta aactggagaa gttacactga agtataatca cactgaatgt
     7141 tagacttttt catctgccaa aaccaaaaac cattttgatc tccctgtggt tatcaatata
     7201 acgcacaatc acaagtgtat gaggacttag aaattaatcc tttgtggtag gagttctgtt
     7261 gaatgatgga aatcttatta ctaccacaag actatttgat ctggtaattg gagacttcgg
     7321 gatttaggag atctccatgt ctgtatttac tctaccactg ctaaagtgtg tggtcctggg
     7381 tagtttactt gcttgcggaa aatgagaatt gatggtgtcc ccaatgcccc acctcacaga
     7441 gttactaaaa aatgtctgta aagcatattt acctcttggg agataggcac tatgtaaata
     7501 aggtaaaatt tctgttatta caattattca taataatatt cttttcttat ttctaagcct
     7561 ttctgggaaa tcatttcagt ccacaccaac catattattc agggttcctg ccatatgtgt
     7621 ggggtatcct actgatacac acgtattcaa agtttatggg tacaacaaag acatagtaca
     7681 tgtacataat atgtatgtga atatagttaa atatatttct tcacaatatt ttaaactgtg
     7741 aagaacttta tcatacagga aacttaaaac aagaggtgtc aaaagaccca aattaggtgc
     7801 attttacttg tttatgatgg cataaccatt gctttaaaat gtttagacag tagaatattg
     7861 aatttatgct ctatttttgt ttatttaagc aacacttaat gtaaaagtgc aacaggcaat
     7921 tgaatccaa
//
