LOCUS       sequence-396634-        3454 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Contains gRNA for GBX2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 3454)
  AUTHORS   Maimaitili M, Chen M, Febbraro F, Ucuncu E, Kelly R, Niclis JC, 
            Christiansen JR, Mermet-Joret N, Niculescu D, Lauritsen J, Iannielli
            A, Klaestrup IH, Jensen UB, Qvist P, Nabavi S, Broccoli V, Nykjaer 
            A, Romero-Ramos M, Denham M
  TITLE     Enhanced production of mesencephalic dopaminergic neurons from 
            lineage-restricted human undifferentiated stem cells.
  JOURNAL   Nat Commun. 2023 Dec 5;14(1):7871. doi: 10.1038/s41467-023-43471-0.
  PUBMED    38052784
REFERENCE   2  (bases 1 to 3454)
  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: "Nat 
            Commun."; date: "2023-12-5"; volume: "14(1):7871. doi"; pages: " 
            10.1038/s41467-023-43471-0"
COMMENT     Sequence Label: phH1-GBX2-gRNA
FEATURES             Location/Qualifiers
     source          1..3454
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..215
                     /label=H1 promoter
                     /note="human H1 RNA promoter"
     primer_bind     136..155
                     /label=H1
                     /note="Human H1 promoter, forward primer"
     misc_RNA        245..320
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(365..385)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(365..383)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(404..420)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(404..420)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(417..439)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    428..444
                     /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(452..458,459..476,477..482))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    497..518
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(635..652)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(806..1394)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(886..905)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(1471..2265)
                     /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             complement(1471..2265)
                     /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"
     primer_bind     complement(1582..1601)
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     2192..2211
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     promoter        complement(2300..2657)
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      2314..2449
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     enhancer        2466..2657
                     /label=SV40 enhancer
                     /note="enhancer for the SV40 early promoter (Herr, 1993)"
     primer_bind     2614..2634
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        complement(2659..2763)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(2789..3244)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2926..2947)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     3138..3157
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3371..3393
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     3385..3402
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     3386..3402
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3412..3431
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        3412..3430
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
ORIGIN
        1 gaacgctgac gtcatcaacc cgctccaagg aatcgcgggc ccagtgtcac taggcgggaa
       61 cacccagcgc gcgtgcgccc tggcaggaag atggctgtga gggacagggg agtggcgccc
      121 tgcaatattt gcatgtcgct atgtgttctg ggaaatcacc ataaacgtga aatgtctttg
      181 gatttgggaa tcttataagt tctgtatgag accactcttt cccaaaaggt ggaagacgac
      241 ccgagtttta gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa
      301 aagtggcacc gagtcggtgc tttttttggt ctgagacgcc gcggtggagc tccagctttt
      361 gttcccttta gtgagggtta attgcgcgct tggcgtaatc atggtcatag ctgtttcctg
      421 tgtgaaattg ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta
      481 aagcctgggg tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg
      541 ctttccagtc gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga
      601 gaggcggttt gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg
      661 tcgttcggct gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag
      721 aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
      781 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca
      841 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
      901 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc
      961 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc
     1021 tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc
     1081 ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
     1141 tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
     1201 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta
     1261 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca
     1321 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa
     1381 aaaaaggatc tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg
     1441 aaaactcacg ttaagggatt ttggtcatga tcagaagaac tcgtcaagaa ggcgatagaa
     1501 ggcgatgcgc tgcgaatcgg gagcggcgat accgtaaagc acgaggaagc ggtcagccca
     1561 ttcgccgcca agctcttcag caatatcacg ggtagccaac gctatgtcct gatagcggtc
     1621 cgccacaccc agccggccac agtcgatgaa tccagaaaag cggccatttt ccaccatgat
     1681 attcggcaag caggcatcgc catgggtcac gacgagatcc tcgccgtcgg gcatgctcgc
     1741 cttgagcctg gcgaacagtt cggctggcgc gagcccctga tgctcttcgt ccagatcatc
     1801 ctgatcgaca agaccggctt ccatccgagt acgtgctcgc tcgatgcgat gtttcgcttg
     1861 gtggtcgaat gggcaggtag ccggatcaag cgtatgcagc cgccgcattg catcagccat
     1921 gatggatact ttctcggcag gagcaaggtg agatgacagg agatcctgcc ccggcacttc
     1981 gcccaatagc agccagtccc ttcccgcttc agtgacaacg tcgagcacag ctgcgcaagg
     2041 aacgcccgtc gtggccagcc acgatagccg cgctgcctcg tcttgcagtt cattcagggc
     2101 accggacagg tcggtcttga caaaaagaac cgggcgcccc tgcgctgaca gccggaacac
     2161 ggcggcatca gagcagccga ttgtctgttg tgcccagtca tagccgaata gcctctccac
     2221 ccaagcggcc ggagaacctg cgtgcaatcc atcttgttca atcatgcgaa acgatcctca
     2281 tcctgtctct tgatcgatct ttgcaaaagc ctaggcctcc aaaaaagcct cctcactact
     2341 tctggaatag ctcagaggcc gaggcggcct cggcctctgc ataaataaaa aaaattagtc
     2401 agccatgggg cggagaatgg gcggaactgg gcggagttag gggcgggatg ggcggagtta
     2461 ggggcgggac tatggttgct gactaattga gatgcatgct ttgcatactt ctgcctgctg
     2521 gggagcctgg ggactttcca cacctggttg ctgactaatt gagatgcatg ctttgcatac
     2581 ttctgcctgc tggggagcct ggggactttc cacaccctaa ctgacacaca ttccacagct
     2641 ggttctttcc gcctcaggac tcttcctttt tcaatattat tgaagcattt atcagggtta
     2701 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     2761 gcgcacattt ccccgaaaag tgccacctaa attgtaagcg ttaatatttt gttaaaattc
     2821 gcgttaaatt tttgttaaat cagctcattt tttaaccaat aggccgaaat cggcaaaatc
     2881 ccttataaat caaaagaata gaccgagata gggttgagtg ttgttccagt ttggaacaag
     2941 agtccactat taaagaacgt ggactccaac gtcaaagggc gaaaaaccgt ctatcagggc
     3001 gatggcccac tacgtgaacc atcaccctaa tcaagttttt tggggtcgag gtgccgtaaa
     3061 gcactaaatc ggaaccctaa agggagcccc cgatttagag cttgacgggg aaagccggcg
     3121 aacgtggcga gaaaggaagg gaagaaagcg aaaggagcgg gcgctagggc gctggcaagt
     3181 gtagcggtca cgctgcgcgt aaccaccaca cccgccgcgc ttaatgcgcc gctacagggc
     3241 gcgtcccatt cgccattcag gctgcgcaac tgttgggaag ggcgatcggt gcgggcctct
     3301 tcgctattac gccagctggc gaaaggggga tgtgctgcaa ggcgattaag ttgggtaacg
     3361 ccagggtttt cccagtcacg acgttgtaaa acgacggcca gtgagcgcgc gtaatacgac
     3421 tcactatagg gcgaattggg tacccgtctc acct
//
