LOCUS       sequence-410960-        4966 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  cloning backbone for sgRNA using OptScf2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4966)
  AUTHORS   Haldrup J, Andersen S, Labial ARL, Wolff JH, Frandsen FP, Skov TW, 
            Rovsing AB, Nielsen I, Jakobsen TS, Askou AL, Thomsen MK, Corydon 
            TJ, Thomsen EA, Mikkelsen JG
  TITLE     Engineered lentivirus-derived nanoparticles (LVNPs) for delivery of 
            CRISPR/Cas ribonucleoprotein complexes supporting base editing, 
            prime editing and in vivo gene modification.
  JOURNAL   Nucleic Acids Res. 2023 Sep 8:gkad676. doi: 10.1093/nar/gkad676.
  PUBMED    37678882
REFERENCE   2  (bases 1 to 4966)
  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: "Nucleic 
            Acids Res. 2023 Sep 8:gkad676. doi: 10.1093/nar/gkad676."
COMMENT     Sequence Label: pU6-sgRNA-CBh-eGFP
FEATURES             Location/Qualifiers
     source          1..4966
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        1..249
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     1..21
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     172..191
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     enhancer        460..745
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer; 
                     contains an 18-bp deletion relative to the standard CMV 
                     enhancer"
     intron          1025..1252
                     /label=hybrid intron
                     /note="hybrid between chicken beta-actin (CBA) and minute 
                     virus of mice (MMV) introns (Gray et al., 2011)"
     regulatory      1265..1274
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(1271..1273,1274..1276,1277..1987)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(1271..1273,1274..1276,1277..1987)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(1316..1337)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1577..1596)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1924..1945
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2007..2024)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2013..2220
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   2229..2369
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   2229..2358
                     /label=AAV2 ITR
     rep_origin      2444..2899
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2531..2550)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     2741..2762
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(2916..2935)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3035..3057
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3095..3113)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3181..3285
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3286..3354,3355..4146)
                     /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(3504..3523)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4317..4905
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4806..4825
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 gagggcctat ttcccatgat tccttcatat ttgcatatac gatacaaggc tgttagagag
       61 ataattggaa ttaatttgac tgtaaacaca aagatattag tacaaaatac gtgacgtaga
      121 aagtaataat ttcttgggta gtttgcagtt ttaaaattat gttttaaaat ggactatcat
      181 atgcttaccg taacttgaaa gtatttcgat ttcttggctt tatatatctt gtggaaagga
      241 cgaaacaccg ggtcttcgag aagacctgtt taagagctat gctggaaaca gcatagcaag
      301 tttaaataag gctagtccgt tatcaacttt gctggaaaca gcaaagtggc accgagtcgg
      361 tgcttttttg ttttagagct agaaatagca agttaaaata aggctagtcc gtttttagcg
      421 cgtgcgccaa ttctgcagac aaatggctct agaggtaccc gttacataac ttacggtaaa
      481 tggcccgcct ggctgaccgc ccaacgaccc ccgcccattg acgtcaatag taacgccaat
      541 agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc acttggcagt
      601 acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg gtaaatggcc
      661 cgcctggcat tgtgcccagt acatgacctt atgggacttt cctacttggc agtacatcta
      721 cgtattagtc atcgctatta ccatggtcga ggtgagcccc acgttctgct tcactctccc
      781 catctccccc ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc
      841 agcgatgggg gcgggggggg ggggggggsg sgcgcsgggg gggggggggg gggggggggg
      901 ggggggsggg gsgrggcgga gaggtgcggc ggcagccaat cagagcggcg cgctccgaaa
      961 gtttcctttt atggcgaggc ggcggcggcg gcggccctat aaaaagcgaa gcgcgcggcg
     1021 ggcgggagtc gctgcgcgct gccttcgccc cgtgccccgc tccgccgccg cctcgcgccg
     1081 cccgccccgg ctctgactga ccgcgttact cccacaggtg agcgggcggg acggcccttc
     1141 tcctccgggc tgtaattagc tgagcaagag gtaagggttt aagggatggt tggttggtgg
     1201 ggtattaatg tttaattacc tggagcacct gcctgaaatc actttttttc aggttggacc
     1261 ggtcgccacc atggtgagca agggcgagga gctgttcacc ggggtggtgc ccatcctggt
     1321 cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg gcgagggcga
     1381 tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc tgcccgtgcc
     1441 ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc gctaccccga
     1501 ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg tccaggagcg
     1561 caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga agttcgaggg
     1621 cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg acggcaacat
     1681 cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca tggccgacaa
     1741 gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg acggcagcgt
     1801 gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg tgctgctgcc
     1861 cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg agaagcgcga
     1921 tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca tggacgagct
     1981 gtacaagcct agagctcgct gatcagcctc gactgtgcct tctagttgcc agccatctgt
     2041 tgtttgcccc tcccccgtgc cttccttgac cctggaaggt gccactccca ctgtcctttc
     2101 ctaataaaat gaggaaattg catcgcattg tctgagtagg tgtcattcta ttctgggggg
     2161 tggggtgggg caggacagca agggggagga ttgggaagag aatagcaggc atgctgggga
     2221 gcggccgcag gaacccctag tgatggagtt ggccactccc tctctgcgcg ctcgctcgct
     2281 cactgaggcc gggcgaccaa aggtcgcccg acgcccgggc tttgcccggg cggcctcagt
     2341 gagcgagcga gcgcgcagct gcctgcaggg gcgcctgatg cggtattttc tccttacgca
     2401 tctgtgcggt atttcacacc gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc
     2461 gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc
     2521 ttagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc
     2581 cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt acggcacctc
     2641 gaccccaaaa aacttgattt gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg
     2701 gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact
     2761 ggaacaacac tcaactctat ctcgggctat tcttttgatt tataagggat tttgccgatt
     2821 tcggtctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa
     2881 atattaacgt ttacaatttt atggtgcact ctcagtacaa tctgctctga tgccgcatag
     2941 ttaagccagc cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc
     3001 ccggcatccg cttacagaca agctgtgacc gtctccggga gctgcatgtg tcagaggttt
     3061 tcaccgtcat caccgaaacg cgcgagacga aagggcctcg tgatacgcct atttttatag
     3121 gttaatgtca tgataataat ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg
     3181 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     3241 caataaccct gataaatgct tcaataatat tgaaaaagga agagtatgag tattcaacat
     3301 ttccgtgtcg cccttattcc cttttttgcg gcattttgcc ttcctgtttt tgctcaccca
     3361 gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg gtgcacgagt gggttacatc
     3421 gaactggatc tcaacagcgg taagatcctt gagagttttc gccccgaaga acgttttcca
     3481 atgatgagca cttttaaagt tctgctatgt ggcgcggtat tatcccgtat tgacgccggg
     3541 caagagcaac tcggtcgccg catacactat tctcagaatg acttggttga gtactcacca
     3601 gtcacagaaa agcatcttac ggatggcatg acagtaagag aattatgcag tgctgccata
     3661 accatgagtg ataacactgc ggccaactta cttctgacaa cgatcggagg accgaaggag
     3721 ctaaccgctt ttttgcacaa catgggggat catgtaactc gccttgatcg ttgggaaccg
     3781 gagctgaatg aagccatacc aaacgacgag cgtgacacca cgatgcctgt agcaatggca
     3841 acaacgttgc gcaaactatt aactggcgaa ctacttactc tagcttcccg gcaacaatta
     3901 atagactgga tggaggcgga taaagttgca ggaccacttc tgcgctcggc ccttccggct
     3961 ggctggttta ttgctgataa atctggagcc ggtgagcgtg gaagccgcgg tatcattgca
     4021 gcactggggc cagatggtaa gccctcccgt atcgtagtta tctacacgac ggggagtcag
     4081 gcaactatgg atgaacgaaa tagacagatc gctgagatag gtgcctcact gattaagcat
     4141 tggtaactgt cagaccaagt ttactcatat atactttaga ttgatttaaa acttcatttt
     4201 taatttaaaa ggatctaggt gaagatcctt tttgataatc tcatgaccaa aatcccttaa
     4261 cgtgagtttt cgttccactg agcgtcagac cccgtagaaa agatcaaagg atcttcttga
     4321 gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg
     4381 gtggtttgtt tgccggatca agagctacca actctttttc cgaaggtaac tggcttcagc
     4441 agagcgcaga taccaaatac tgttcttcta gtgtagccgt agttaggcca ccacttcaag
     4501 aactctgtag caccgcctac atacctcgct ctgctaatcc tgttaccagt ggctgctgcc
     4561 agtggcgata agtcgtgtct taccgggttg gactcaagac gatagttacc ggataaggcg
     4621 cagcggtcgg gctgaacggg gggttcgtgc acacagccca gcttggagcg aacgacctac
     4681 accgaactga gatacctaca gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga
     4741 aaggcggaca ggtatccggt aagcggcagg gtcggaacag gagagcgcac gagggagctt
     4801 ccagggggaa acgcctggta tctttatagt cctgtcgggt ttcgccacct ctgacttgag
     4861 cgtcgatttt tgtgatgctc gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg
     4921 gcctttttac ggttcctggc cttttgctgg ccttttgctc acatgt
//
