LOCUS       sequence-160439-        4658 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Expresses gRNA of interest under U6 promoter (cloning by BpiI) and 
            GFP under PGK promoter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4658)
  AUTHORS   Daneshvar K, Pondick JV, Kim BM, Zhou C, York SR, Macklin JA, 
            Abualteen A, Tan B, Sigova AA, Marcho C, Tremblay KD, Mager J, Choi 
            MY, Mullen AC
  TITLE     DIGIT Is a Conserved Long Noncoding RNA that Regulates GSC 
            Expression to Control Definitive Endoderm Differentiation of 
            Embryonic Stem Cells.
  JOURNAL   Cell Rep. 2016 Oct 4;17(2):353-365. doi: 
            10.1016/j.celrep.2016.09.017.
  PUBMED    27705785
REFERENCE   2  (bases 1 to 4658)
  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: 
            "10.1016/j.celrep.2016.09"; journalName: "Cell Rep"; date: 
            "2016-10-4- 4"; volume: "17"; issue: "2"; pages: "353-365"
COMMENT     Sequence Label: pgRGFP
FEATURES             Location/Qualifiers
     source          1..4658
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      627..1215
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     1116..1135
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     promoter        1277..1525
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1277..1297
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     1448..1467
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        1544..1619
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     primer_bind     complement(1761..1784)
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     primer_bind     2123..2142
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     regulatory      2218..2227
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(2224..2226,2227..2229,2230..2943)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2224..2226,2227..2229,2230..2940)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(2269..2290)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2530..2549)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2877..2898
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2975..2992)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2981..3188
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3197..3337
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3197..3326
                     /label=AAV2 ITR
     rep_origin      3412..3867
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3499..3518)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3709..3730
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3884..3903)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4003..4025
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(4063..4081)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4149..4253
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4254..4322,4323..4658,1..456)
                     /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(4472..4491)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
       61 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
      121 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
      181 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
      241 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
      301 ggtgagcgtg gaagccgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
      361 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
      421 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
      481 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
      541 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
      601 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
      661 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
      721 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta
      781 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
      841 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
      901 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
      961 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     1021 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     1081 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     1141 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     1201 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     1261 ccttttgctc acatgtgagg gcctatttcc catgattcct tcatatttgc atatacgata
     1321 caaggctgtt agagagataa ttggaattaa tttgactgta aacacaaaga tattagtaca
     1381 aaatacgtga cgtagaaagt aataatttct tgggtagttt gcagttttaa aattatgttt
     1441 taaaatggac tatcatatgc ttaccgtaac ttgaaagtat ttcgatttct tggctttata
     1501 tatcttgtgg aaaggacgaa acaccgggtc ttcgagaaga cctgttttag agctagaaat
     1561 agcaagttaa aataaggcta gtccgttatc aacttgaaaa agtggcaccg agtcggtgct
     1621 tttttgtttt agagctagaa atagcaagtt aaaataaggc tagtccgttt ttagcgcgtg
     1681 cgccaattct gcagacaaat ggctcgtcga cctcgaaatt ctaccgggta ggggaggcgc
     1741 ttttcccaag gcagtctgga gcatgcgctt tagcagcccc gctgggcact tggcgctaca
     1801 caagtggcct ctggcctcgc acacattcca catccaccgg taggcgccaa ccggctccgt
     1861 tctttggtgg ccccttcgcg ccaccttcta ctcctcccct agtcaggaag ttcccccccg
     1921 ccccgcagct cgcgtcgtgc aggacgtgac aaatggaagt agcacgtctc actagtctcg
     1981 tgcagatgga cagcaccgct gagcaatgga agcgggtagg cctttggggc agcggccaat
     2041 agcagctttg ctccttcgct ttctgggctc aggggcgggc tcaggggcgg ggcgggcgcc
     2101 cgaaggtcct ccggaggccc ggcattctgc acgcttcaaa agcgcacgtc tgccgcgctg
     2161 ttctcctctt cctcatctcc gggcctttcg acctgcatcc atctagatct cccggtcgcc
     2221 accatggtga gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg
     2281 gacggcgacg taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc
     2341 tacggcaagc tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc
     2401 accctcgtga ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg
     2461 aagcagcacg acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc
     2521 ttcttcaagg acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc
     2581 ctggtgaacc gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg
     2641 cacaagctgg agtacaacta caacagccac aacgtctata tcatggccga caagcagaag
     2701 aacggcatca aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc
     2761 gccgaccact accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac
     2821 cactacctga gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg
     2881 gtcctgctgg agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag
     2941 tagcggccgc ataaccctag agctcgctga tcagcctcga ctgtgccttc tagttgccag
     3001 ccatctgttg tttgcccctc ccccgtgcct tccttgaccc tggaaggtgc cactcccact
     3061 gtcctttcct aataaaatga ggaaattgca tcgcattgtc tgagtaggtg tcattctatt
     3121 ctggggggtg gggtggggca ggacagcaag ggggaggatt gggaagagaa tagcaggcat
     3181 gctggggagc ggccgcagga acccctagtg atggagttgg ccactccctc tctgcgcgct
     3241 cgctcgctca ctgaggccgg gcgaccaaag gtcgcccgac gcccgggctt tgcccgggcg
     3301 gcctcagtga gcgagcgagc gcgcagctgc ctgcaggggc gcctgatgcg gtattttctc
     3361 cttacgcatc tgtgcggtat ttcacaccgc atacgtcaaa gcaaccatag tacgcgccct
     3421 gtagcggcgc attaagcgcg gcgggtgtgg tggttacgcg cagcgtgacc gctacacttg
     3481 ccagcgccct agcgcccgct cctttcgctt tcttcccttc ctttctcgcc acgttcgccg
     3541 gctttccccg tcaagctcta aatcgggggc tccctttagg gttccgattt agtgctttac
     3601 ggcacctcga ccccaaaaaa cttgatttgg gtgatggttc acgtagtggg ccatcgccct
     3661 gatagacggt ttttcgccct ttgacgttgg agtccacgtt ctttaatagt ggactcttgt
     3721 tccaaactgg aacaacactc aaccctatct cgggctattc ttttgattta taagggattt
     3781 tgccgatttc ggcctattgg ttaaaaaatg agctgattta acaaaaattt aacgcgaatt
     3841 ttaacaaaat attaacgttt acaattttat ggtgcactct cagtacaatc tgctctgatg
     3901 ccgcatagtt aagccagccc cgacacccgc caacacccgc tgacgcgccc tgacgggctt
     3961 gtctgctccc ggcatccgct tacagacaag ctgtgaccgt ctccgggagc tgcatgtgtc
     4021 agaggttttc accgtcatca ccgaaacgcg cgagacgaaa gggcctcgtg atacgcctat
     4081 ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc acttttcggg
     4141 gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat atgtatccgc
     4201 tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag agtatgagta
     4261 ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt cctgtttttg
     4321 ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt gcacgagtgg
     4381 gttacatcga actggatctc aacagcggta agatccttga gagttttcgc cccgaagaac
     4441 gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta tcccgtattg
     4501 acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac ttggttgagt
     4561 actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa ttatgcagtg
     4621 ctgccataac catgagtgat aacactgcgg ccaactta
//
