LOCUS       sequence-211705-        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      1896..1905
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             1902..2276
                     /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      2414..3002
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2903..2922
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3156..3173
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3290..3311
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3326..3331,3332..3349,3350..3356)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3364..3380
                     /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     3369..3391
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3388..3404
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3388..3404
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        3422..3440
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     3422..3439
                     /label=SP6
                     /note="SP6 promoter, forward primer"
     CDS             4479..4499
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     CDS             4512..4556
                     /codon_start=1
                     /product="affinity and epitope tag derived from pancreatic 
                     ribonuclease A"
                     /label=S-Tag
                     /translation="KETAAAKFERQHMDS"
     CDS             4596..4619
                     /codon_start=1
                     /product="recognition and cleavage site for human 
                     rhinovirus 3C and PreScission proteases"
                     /label=HRV 3C site
                     /translation="LEVLFQGP"
     CDS             join(4647..4649,4650..4652,4653..5366)
                     /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(4692..4713)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(4953..4972)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     misc_feature    join(5404..5977,5978..5980,5981..5989)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(5570..5587)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     5797..5816
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             6071..6874
                     /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 ctcgccgcca
      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 ccgcatacag gtggcacttt tcggggaaat gtgcgcggaa
     1801 cccctatttg tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac
     1861 cctgataaat gcttcaataa tagcacgtga ggagggccac catggccaag ttgaccagtg
     1921 ccgttccggt gctcaccgcg cgcgacgtcg ccggagcggt cgagttctgg accgaccggc
     1981 tcgggttctc ccgggacttc gtggaggacg acttcgccgg tgtggtccgg gacgacgtga
     2041 ccctgttcat cagcgcggtc caggaccagg tggtgccgga caacaccctg gcctgggtgt
     2101 gggtgcgcgg cctggacgag ctgtacgccg agtggtcgga ggtcgtgtcc acgaacttcc
     2161 gggacgcctc cgggccggcc atgaccgaga tcggcgagca gccgtggggg cgggagttcg
     2221 ccctgcgcga cccggccggc aactgcgtgc acttcgtggc cgaggagcag gactgacacg
     2281 tgctaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     2341 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     2401 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     2461 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     2521 aggtaactgg cttcagcaga gcgcagatac caaatactgt ccttctagtg tagccgtagt
     2581 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     2641 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     2701 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     2761 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     2821 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     2881 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     2941 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     3001 aaaacgccag caacgcggcc tttttacggt tcctgggctt ttgctggcct tttgctcaca
     3061 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     3121 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     3181 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     3241 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     3301 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     3361 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     3421 tatttaggtg acactataga atactcaagc tatgcatcaa gcttggtacc gagctcggat
     3481 ccactagtaa cggccgccag tgtgctggaa ttcgcccttt cggcaagatt gagttccttg
     3541 caagctgttg gactgagcac ctcagttgct tgctgactgt tggctagagc ccactcttag
     3601 ttccttgcca tgtgggcctc tccacagggt ggctcacagc atggcagcag cttttctcag
     3661 agtaagtgag caagagcgta cctaagatgg aggccacagt ttcttgtaag ctaatctcag
     3721 aacggacatc ctatcaattc tgccatattt tttttcatta gaagttagta agtctaggtc
     3781 cacttagtag gaggaggttg cgcaagggtg cgaataccag gatgaaggga tcattggagg
     3841 ccattttagg ggctgcctac cacacatggt gacatacaaa tgggagtgta ttccatatgt
     3901 attgaaaata tgtttatata ctacattatt ttattttgct aattagaagc agttgaaatc
     3961 agatgacatc taattttaaa aagtcagaac ctctatgttc ttgtgaataa aaattcttaa
     4021 cagtaaagaa ttttgattga tcaaatgttt tatgtgatat atatgcttta taatatagta
     4081 actagctaac gatttgttac tgagattttg agaaatttgt caacttttgc ctttattcag
     4141 atgtataaaa ccttgctttc tattcgttag tgacactgat aagcaagaat tttatttata
     4201 tttaatgtta ataatttatc ctgtttttcc tatttcccta ttcctgatct ctcttagatc
     4261 gatctggaat agaaaacgtc aactctgtgg aggctttgca ggaaactctc attcgtgcac
     4321 taaggacctt aataatgaaa aaccatccaa atgaggcctc tatttttaca aaactgcttc
     4381 taaagttgcc agatcttcga tctttaaaca acatgcactc tgaggagctc ttggccttta
     4441 aagttcaccc tgattatgat attccaacta ctgcaagcga gaatctttat tttcagggcg
     4501 ccgccaaatt caaagaaacc gctgctgcta aattcgaacg ccagcacatg gacagcggag
     4561 gtggaggttc gagcggtccc tcgggttcgt cgagcctgga agttctgttc caggggcccc
     4621 tgtcgtcgag cggtccctcg ggttcgatgg tgagcaaagg cgaggagctg ttcaccgggg
     4681 tggtgcccat cctggtcgag ctggacggcg acgtaaacgg ccacaagttc agcgtgtccg
     4741 gcgagggcga gggcgatgcc acctacggca agctgaccct gaagttcatc tgcaccaccg
     4801 gcaagctgcc cgtgccctgg cccaccctcg tgaccaccct gacctacggc gtgcagtgct
     4861 tcagccgcta ccccgaccac atgaagcagc acgacttctt caagtccgcc atgcccgaag
     4921 gctacgtcca ggagcgcacc atcttcttca aggacgacgg caactacaag acccgcgccg
     4981 aggtgaagtt cgagggcgac accctggtga accgcatcga gctgaagggc atcgacttca
     5041 aggaggacgg caacatcctg gggcacaagc tggagtacaa ctacaacagc cacaacgtct
     5101 atatcatggc cgacaagcag aagaacggca tcaaggtgaa cttcaagatc cgccacaaca
     5161 tcgaggacgg cagcgtgcag ctcgccgacc actaccagca gaacaccccc atcggcgacg
     5221 gccccgtgct gctgcccgac aaccactacc tgagcaccca gtccgccctg agcaaagacc
     5281 ccaacgagaa gcgcgatcac atggtcctta aggagttcgt gaccgccgcc gggatcactc
     5341 tcggcatgga cgagctgtac aagtaatgat ctagagtcga gttaattaag aattccgccc
     5401 ccccccctct ccctcccccc ccctaacgtt actggccgaa gccgcttgga ataaggccgg
     5461 tgtgcgtttg tctatatgtt attttccacc atattgccgt cttttggcaa tgtgagggcc
     5521 cggaaacctg gccctgtctt cttgacgagc attcctaggg gtctttcccc tctcgccaaa
     5581 ggaatgcagg gtctgttgaa tgtcgtgaag gaagcagttc ctctggaagc ttcttgaaga
     5641 caaacaacgt ctgtagcgac cctttgcagg cagcggaacc ccccacctgg cgacaggtgc
     5701 ctctgcggcc aaaagccacg tgtataagat acacctgcaa aggcggcaca accccagtgc
     5761 cacgttgtga gttggatagt tgtggaaaga gtcaaatggc tctcctcaag cgtattcaac
     5821 aaggggctga aggatgccca gaaggtaccc cattgtatgg gatctgatct ggggcctcgg
     5881 tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac gtctaggccc cccgaaccac
     5941 ggggacgtgg ttttcctttg aaaaacacga tgataatatg gccacaacct gttacattgc
     6001 acaagataaa aatatatcat cacgaacagt aaaactgtct gcttacataa acagtaatac
     6061 aaggggtgtt atgggatcgg ccattgaaca agatggattg cacgcaggtt ctccggccgc
     6121 ttgggtggag aggctattcg gctatgactg ggcacaacag acaatcggct gctctgatgc
     6181 cgccgtgttc cggctgtcag cgcaggggcg cccggttctt tttgtcaaga ccgacctgtc
     6241 cggtgccctg aatgaactgc aggacgaggc agcgcggcta tcgtggctgg ccacgacggg
     6301 cgttccttgc gcagctgtgc tcgacgttgt cactgaagcg ggaagggact ggctgctatt
     6361 gggcgaagtg ccggggcagg atctcctgtc atctcacctt gctcctgccg agaaagtatc
     6421 catcatggct gatgcaatgc ggcggctgca tacgcttgat ccggctacct gcccattcga
     6481 ccaccaagcg aaacatcgca tcgagcgagc acgtactcgg atggaagccg gtcttgtcga
     6541 tcaggatgat ctggacgaag agcatcaggg gctcgcgcca gccgaactgt tcgccaggct
     6601 caaggcgcgc atgcccgacg gcgaggatct cgtcgtgacc catggcgatg cctgcttgcc
     6661 gaatatcatg gtggaaaatg gccgcttttc tggattcatc gactgtggcc ggctgggtgt
     6721 ggcggaccgc tatcaggaca tagcgttggc tacccgtgat attgctgaag agcttggcgg
     6781 cgaatgggct gaccgcttcc tcgtgcttta cggtatcgcc gctcccgatt cgcagcgcat
     6841 cgccttctat cgccttcttg acgagttctt ctgaggcctt 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
//
