LOCUS       sequence-472877-        4027 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  HITI insert construct with Ckm gRNA and GFP transgene.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4027)
  AUTHORS   Padmaswari MH, Bulliard G, Agrawal S, Jia MS, Khadgi S, Murach KA, 
            Nelson CE
  TITLE     Precision and efficacy of RNA-guided DNA integration in 
            high-expressing muscle loci.
  JOURNAL   Mol Ther Nucleic Acids. 2024 Sep 2;35(4):102320. doi: 
            10.1016/j.omtn.2024.102320. eCollection 2024 Dec 10.
  PUBMED    39398225
REFERENCE   2  (bases 1 to 4027)
  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: "Mol Ther 
            Nucleic Acids."; date: "2024-09-2"; volume: "35(4):102320. doi"; 
            pages: " 10.1016/j.omtn.2024.102320. eCollection 2024 Dec 10"
COMMENT     Sequence Label: p-HITI-GFP-Ckm
FEATURES             Location/Qualifiers
     source          1..4027
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     polyA_signal    1..122
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(38..57)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     92..111
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(276..295)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(277..295)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(305..322)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(305..321)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(314..336)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      463..918
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(550..569)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     760..781
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        944..1048
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1049..1117,1118..1909)
                     /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(1267..1286)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2080..2668
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     2569..2588
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     2822..2839
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    2956..2977
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(2992..2997,2998..3015,3016..3022)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3030..3046
                     /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     3035..3057
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3054..3070
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3054..3070
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     3089..3109
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        3091..3109
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     regulatory      3276..3285
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(3282..3284,3285..3287,3288..4001)
                     /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(3282..3284,3285..3287,3288..3998)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(3327..3348)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(3588..3607)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     3935..3956
                     /label=EGFP-C
                     /note="EGFP, forward primer"
ORIGIN
        1 aacttgttta ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca
       61 aataaagcat ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct
      121 tagtcaggct gcccctccgc agagggagcc ggctcggggt ccccgcgtaa gccagcctgg
      181 tgccacccgg agcggcgtta acgggtgtgt ggtgtctcgg ctctgcaggg cgtcgtcgac
      241 ctatgtgatc ctgctgggga ctggtaccca attcgcccta tagtgagtcg tattacgcgc
      301 gctcactggc cgtcgtttta caacgtcgtg actgggaaaa ccctggcgtt acccaactta
      361 atcgccttgc agcacatccc cctttcgcca gctggcgtaa tagcgaagag gcccgcaccg
      421 atcgcccttc ccaacagttg cgcagcctga atggcgaatg ggacgcgccc tgtagcggcg
      481 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc
      541 tagcgcccgc tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc
      601 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
      661 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg
      721 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg
      781 gaacaacact caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt
      841 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat tttaacaaaa
      901 tattaacgct tacaatttag gtggcacttt tcggggaaat gtgcgcggaa cccctatttg
      961 tttatttttc taaatacatt caaatatgta tccgctcatg agacaataac cctgataaat
     1021 gcttcaataa tattgaaaaa ggaagagtat gagtattcaa catttccgtg tcgcccttat
     1081 tccctttttt gcggcatttt gccttcctgt ttttgctcac ccagaaacgc tggtgaaagt
     1141 aaaagatgct gaagatcagt tgggtgcacg agtgggttac atcgaactgg atctcaacag
     1201 cggtaagatc cttgagagtt ttcgccccga agaacgtttt ccaatgatga gcacttttaa
     1261 agttctgcta tgtggcgcgg tattatcccg tattgacgcc gggcaagagc aactcggtcg
     1321 ccgcatacac tattctcaga atgacttggt tgagtactca ccagtcacag aaaagcatct
     1381 tacggatggc atgacagtaa gagaattatg cagtgctgcc ataaccatga gtgataacac
     1441 tgcggccaac ttacttctga caacgatcgg aggaccgaag gagctaaccg cttttttgca
     1501 caacatgggg gatcatgtaa ctcgccttga tcgttgggaa ccggagctga atgaagccat
     1561 accaaacgac gagcgtgaca ccacgatgcc tgtagcaatg gcaacaacgt tgcgcaaact
     1621 attaactggc gaactactta ctctagcttc ccggcaacaa ttaatagact ggatggaggc
     1681 ggataaagtt gcaggaccac ttctgcgctc ggcccttccg gctggctggt ttattgctga
     1741 taaatctgga gccggtgagc gtgggtctcg cggtatcatt gcagcactgg ggccagatgg
     1801 taagccctcc cgtatcgtag ttatctacac gacggggagt caggcaacta tggatgaacg
     1861 aaatagacag atcgctgaga taggtgcctc actgattaag cattggtaac tgtcagacca
     1921 agtttactca tatatacttt agattgattt aaaacttcat ttttaattta aaaggatcta
     1981 ggtgaagatc ctttttgata atctcatgac caaaatccct taacgtgagt tttcgttcca
     2041 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     2101 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     2161 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     2221 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     2281 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     2341 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     2401 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     2461 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     2521 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     2581 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     2641 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     2701 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     2761 taaccgtatt accgcctttg agtgagctga taccgctcgc cgcagccgaa cgaccgagcg
     2821 cagcgagtca gtgagcgagg aagcggaaga gcgcccaata cgcaaaccgc ctctccccgc
     2881 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     2941 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     3001 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     3061 cagctatgac catgattacg ccaagcgcgc aattaaccct cactaaaggg aacaaaagct
     3121 ggagctcccc tatgtgatcc tgctggggac tatgtcccta ggttcctggg gagggaggac
     3181 gtcggggcca gccaggctta cccccccctg ccgctgagac ttctgcgctg atgcaccgcg
     3241 cctcttcgcg gtctccctgt ccttgcagac cggtcgccac catggtgagc aagggcgagg
     3301 agctgttcac cggggtggtg cccatcctgg tcgagctgga cggcgacgta aacggccaca
     3361 agttcagcgt gtccggcgag ggcgagggcg atgccaccta cggcaagctg accctgaagt
     3421 tcatctgcac caccggcaag ctgcccgtgc cctggcccac cctcgtgacc accctgacct
     3481 acggcgtgca gtgcttcagc cgctaccccg accacatgaa gcagcacgac ttcttcaagt
     3541 ccgccatgcc cgaaggctac gtccaggagc gcaccatctt cttcaaggac gacggcaact
     3601 acaagacccg cgccgaggtg aagttcgagg gcgacaccct ggtgaaccgc atcgagctga
     3661 agggcatcga cttcaaggag gacggcaaca tcctggggca caagctggag tacaactaca
     3721 acagccacaa cgtctatatc atggccgaca agcagaagaa cggcatcaag gtgaacttca
     3781 agatccgcca caacatcgag gacggcagcg tgcagctcgc cgaccactac cagcagaaca
     3841 cccccatcgg cgacggcccc gtgctgctgc ccgacaacca ctacctgagc acccagtccg
     3901 ccctgagcaa agaccccaac gagaagcgcg atcacatggt cctgctggag ttcgtgaccg
     3961 ccgccgggat cactctcggc atggacgagc tgtacaagta atcgtttaga ccgctgaagg
     4021 atccggc
//
