LOCUS       sequence-322360-        6615 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  donor plasmid for FMR1-Nluc.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6615)
  AUTHORS   Li M, Zhao H, Ananiev GE, Musser MT, Ness KH, Maglaque DL, Saha K, 
            Bhattacharyya A, Zhao X
  TITLE     Establishment of Reporter Lines for Detecting Fragile X Mental 
            Retardation (FMR1) Gene Reactivation in Human Neural Cells.
  JOURNAL   Stem Cells. 2017 Jan;35(1):158-169. doi: 10.1002/stem.2463. Epub 
            2016 Jul 27.
  PUBMED    27422057
REFERENCE   2  (bases 1 to 6615)
  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: "10.1002/stem.2463";
            journalName: "Stem Cells"; date: "2017-01"; volume: "35"; issue: 
            "1"; pages: "158-169"
COMMENT     Sequence Label: FMR1-E17B-P2A-NLuc
FEATURES             Location/Qualifiers
     source          1..6615
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     58..77
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     CDS             join(206..274,275..1066)
                     /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     complement(424..443)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      1237..1825
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1726..1745
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     1979..1996
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2113..2134
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2149..2154,2155..2172,2173..2179)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2187..2203
                     /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     2192..2214
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     2211..2227
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     2211..2227
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             3335..3391
                     /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             3392..3907
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     primer_bind     complement(5054..5073)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5055..5073)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5080..5097)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5080..5096)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5089..5111)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5237..5665
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5324..5343)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5534..5555
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     CDS             join(6009..6615,1..188)
                     /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
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6063..6082)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
ORIGIN
        1 tttctggatt catcgactgt ggccggctgg gtgtggcgga ccgctatcag gacatagcgt
       61 tggctacccg tgatattgct gaagagcttg gcggcgaatg ggctgaccgc ttcctcgtgc
      121 tttacggtat cgccgctccc gattcgcagc gcatcgcctt ctatcgcctt cttgacgagt
      181 tcttctgaat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc
      241 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa
      301 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg
      361 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt
      421 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg
      481 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac
      541 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc
      601 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa
      661 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc
      721 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt
      781 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga
      841 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa
      901 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa
      961 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa
     1021 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt
     1081 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt
     1141 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg
     1201 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt
     1261 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca
     1321 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac
     1381 tgttcttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac
     1441 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct
     1501 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg
     1561 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca
     1621 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt
     1681 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta
     1741 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc
     1801 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc
     1861 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa
     1921 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag
     1981 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg
     2041 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga
     2101 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat
     2161 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag
     2221 ctatgaccat gattacgcca agcttggtac cgagctcgga tccacatgca catacccact
     2281 acttaaacat actttgaaat tacaaccatt tggggatgtt tttagcattt gtgcttgaag
     2341 tagatcaata tttgtagttg ttttagttcc atttgtcact gttaactttc atttgtacct
     2401 ctggaattag cagtgctgta ttcagcattg gcacttaaaa tattttatag ctcttagaac
     2461 actatttttt aaattacatt gatctttata ctctcctagg attccagtcc ttccacaaat
     2521 tattatatgc agttttcttt atactgttcc attcttcaga cagagttcag gttgtaggat
     2581 catctgaatt ttcttagtga aatacaagcc agtagaaacc aaagctcgtg tttttctact
     2641 gtatgactat gggagctctc agtatagaga aacttgatca tacctgtgat agatataaag
     2701 atgtaaatta aagtctagtt agtgagttat ttgagggtaa agccagaata ggaaaacatt
     2761 ttgtttctct tttcctgggc ttcacaatct ttggagaata actaatttta ttttaactct
     2821 agtgttagag gcatgtatgt cttctggtaa tcagtaatag taattcattt tacctgtttg
     2881 tttctcactg ttttattcta cttagaagaa gacatgatag gattgtgagt ttttctctaa
     2941 ctttggatgc agtgagatga ccagtgtgtt ccagttaaag aagaagagtg ttttaaaatc
     3001 ataaaccaaa taaagaatcc taccttacat taatttcttg cacttcttct gttctcatct
     3061 ccatttctct ttttaacatg tggaatttac acttcaggtt taaatttctt gtcaggccaa
     3121 ttacagatta cagtaggata tggtctgtgt atataacaac tataacttgt tttagatcag
     3181 agttgactgc aataatgaaa ggagtgtcca cactaaaaca ttacagaata cctccagtga
     3241 aggtagtcgg ctgcgcacgg gtaaagatcg taaccagaag aaagagaagc cagacagcgt
     3301 ggatggtcag caacctctag ttaacggagt acccgccact aacttctccc tgttgaaaca
     3361 agcaggggat gtcgaagaga atcctggtcc aatggtcttc acactcgaag atttcgttgg
     3421 ggactggcga cagacagccg gctacaacct ggaccaagtc cttgaacagg gaggtgtgtc
     3481 cagtttgttt cagaatctcg gggtgtccgt aactccgatc caaaggattg tcctgagcgg
     3541 tgaaaatggg ctgaagatcg acatccatgt catcatcccg tatgaaggtc tgagcggcga
     3601 ccaaatgggc cagatcgaaa aaatttttaa ggtggtgtac cctgtggatg atcatcactt
     3661 taaggtgatc ctgcactatg gcacactggt aatcgacggg gttacgccga acatgatcga
     3721 ctatttcgga cggccgtatg aaggcatcgc cgtgttcgac ggcaaaaaga tcactgtaac
     3781 agggaccctg tggaacggca acaaaattat cgacgagcgc ctgatcaacc ccgacggctc
     3841 cctgctgttc cgagtaacca tcaacggagt gaccggctgg cggctgtgcg aacgcattct
     3901 ggcgtaagtg aatggagtac cctaaactgc ataattctga agttatattt cctataccat
     3961 ttccgtaatt cttattccat attagaaaac tttgttaggc caaagacaaa tagtaggcaa
     4021 gatggcacag ggcatgaaat gaacacaaat tatgctaaga attttttatt ttttggtatt
     4081 ggccataagc aacaattttc agatttgcac aaaaagatac cttaaaattt gaaacattgc
     4141 ttttaaaact acttagcact tcagggcaga ttttagtttt attttctaaa gtactgagca
     4201 gtgatattct ttgttaattt ggaccatttt cctgcattgg gtgatcattc accagtacat
     4261 tctcagtttt tcttaatata tagcatttat ggtaatcata ttagacttct gttttcaatc
     4321 tcgtatagaa gtcttcatga aatgctatgt catttcatgt cctgtgtcag tttatgtttt
     4381 ggtccacttt tccagtattt tagtggaccc tgaaatgtgt gtgatgtgac atttgtcatt
     4441 ttcattagca aaaaaagttg tatgatctgt gcctttttta tatcttggca ggtaggaata
     4501 ttatatttgg atgcagagtt cagggaagat aagttggaaa cactaaatgt taaagatgta
     4561 gcaaaccctg tcaaacatta gtactttata gaagaatgca tgctttccat atttttttcc
     4621 ttacataaac atcaggttag gcagtataaa gaataggact tgtttttgtt tttgttttgt
     4681 tgcactgaag tttgataaat agtgttattg agagagatgt gtaatttttc tgtatagaca
     4741 ggagaagaaa gaactatctt catctgagag aggctaaaat gttttcagct aggaacaaat
     4801 cttcctggtc gaaagttagt aggatatgcc tgctctttgg cctgatgacc aattttaact
     4861 tagagctttt ttttttaatt ttgtctgccc caagttttgt gaaatttttc atattttaat
     4921 ttcaagctta ttttggagag ataggaaggt catttccatg tatgcataat aatcctgcaa
     4981 agtacaggta ctttgtctaa gaaacattgg aagcaggtgg ccgctcgagc atgcatctag
     5041 agggcccaat tcgccctata gtgagtcgta ttacaattca ctggccgtcg ttttacaacg
     5101 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     5161 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     5221 cctgaatggc gaatggacgc gccctgtagc ggcgcattaa gcgcggcggg tgtggtggtt
     5281 acgcgcagcg tgaccgctac acttgccagc gccctagcgc ccgctccttt cgctttcttc
     5341 ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag ctctaaatcg ggggctccct
     5401 ttagggttcc gatttagtgc tttacggcac ctcgacccca aaaaacttga ttagggtgat
     5461 ggttcacgta gtgggccatc gccctgatag acggtttttc gccctttgac gttggagtcc
     5521 acgttcttta atagtggact cttgttccaa actggaacaa cactcaaccc tatctcggtc
     5581 tattcttttg atttataagg gattttgccg atttcggcct attggttaaa aaatgagctg
     5641 atttaacaaa aatttaacgc gaattttaac aaaattcagg gcgcaagggc tgctaaagga
     5701 agcggaacac gtagaaagcc agtccgcaga aacggtgctg accccggatg aatgtcagct
     5761 actgggctat ctggacaagg gaaaacgcaa gcgcaaagag aaagcaggta gcttgcagtg
     5821 ggcttacatg gcgatagcta gactgggcgg ttttatggac agcaagcgaa ccggaattgc
     5881 cagctggggc gccctctggt aaggttggga agccctgcaa agtaaactgg atggctttct
     5941 tgccgccaag gatctgatgg cgcaggggat caagatctga tcaagagaca ggatgaggat
     6001 cgtttcgcat gattgaacaa gatggattgc acgcaggttc tccggccgct tgggtggaga
     6061 ggctattcgg ctatgactgg gcacaacaga caatcggctg ctctgatgcc gccgtgttcc
     6121 ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac cgacctgtcc ggtgccctga
     6181 atgaactgca ggacgaggca gcgcggctat cgtggctggc cacgacgggc gttccttgcg
     6241 cagctgtgct cgacgttgtc actgaagcgg gaagggactg gctgctattg ggcgaagtgc
     6301 cggggcagga tctcctgtca tcccaccttg ctcctgccga gaaagtatcc atcatggctg
     6361 atgcaatgcg gcggctgcat acgcttgatc cggctacctg cccattcgac caccaagcga
     6421 aacatcgcat cgagcgagca cgtactcgga tggaagccgg tcttgtcgat caggatgatc
     6481 tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt cgccaggctc aaggcgcgca
     6541 tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc ctgcttgccg aatatcatgg
     6601 tggaaaatgg ccgct
//
