LOCUS       sequence-466440-        4954 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid for knock-in of T2A-eGFP-SV40pA at the mouse Grb10 
            locus. Used with PX459-sgRNA048_Grb10 sgRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4954)
  AUTHORS   Lucero K, Han S, Huang P, Qiu X, Mazzoni EO, Reinberg D
  TITLE     CTCF-RNA interactions orchestrate cell-specific chromatin loop 
            organization
  JOURNAL   bioRxiv, March 19, 2025
REFERENCE   2  (bases 1 to 4954)
  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: "bioRxiv, 
            March 19, 2025"
COMMENT     Sequence Label: KL157 - pBlueScript II
            SK-Grb10_HA-T2A-eGFP-SV...
FEATURES             Location/Qualifiers
     source          1..4954
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     666..682
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     667..683
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             1249..1302
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             join(1303..1305,1306..2016)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1303..1305,1306..2016)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     primer_bind     complement(1345..1366)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1606..1625)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1953..1974
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    2050..2171
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(2087..2106)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     2141..2160
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2713..2729)
                     /label=SK primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2713..2729)
                     /label=pBluescriptSK
                     /note="For pBluescript vector"
     promoter        complement(2766..2784)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2805..2821)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2805..2821)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2818..2840)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2829..2845
                     /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(2853..2859,2860..2877,2878..2883))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2898..2919
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3036..3053)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3207..3795)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3287..3306)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3966..4757,4758..4826))
                     /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     4589..4608
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4827..4931)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccgggcc
      661 ccccctcgag gtcgacggta tcgataagct ttagcagctg caatagctgt attattctga
      721 gggtgtcttg gactcccctg atgaacaacc caaaagattt ttttcatgct tatttgataa
      781 tatatagctt tttcaggaag cattgccatc ccaaatggaa taacttcatt ttgaactata
      841 catgtagtat gtatacacac agacttaact tgtattgtat gttactaata tgatttcata
      901 gtaatattaa aatactattt ggtttaggag cctgatcatc caaagataac cattttggtg
      961 gaactctata ccctcactga catttttttc ttctcttgcc attttttatg tacagagcat
     1021 ggtcttgggc ttcaagtact aatgaacaac gatattaact cgtccgtgga aagccttaac
     1081 tcagcttgca acatgcagtc tgatactgat actgcaccac ttcttgagga tggccagcat
     1141 gccagcaacc agggagcagc atctagctcc cggggacagc cacaggcgtc cccgaggcag
     1201 aaaatgcaac gctcgcagcc tgtgcacatt ctcgaattcg gcagtggaga gggcagagga
     1261 agtctgctaa catgcggtga cgtcgaggag aatcctggcc cagtgagcaa gggcgaggag
     1321 ctgttcaccg gggtggtgcc catcctggtc gagctggacg gcgacgtaaa cggccacaag
     1381 ttcagcgtgt ccggcgaggg cgagggcgat gccacctacg gcaagctgac cctgaagttc
     1441 atctgcacca ccggcaagct gcccgtgccc tggcccaccc tcgtgaccac cctgacctac
     1501 ggcgtgcagt gcttcagccg ctaccccgac cacatgaagc agcacgactt cttcaagtcc
     1561 gccatgcccg aaggctacgt ccaggagcgc accatcttct tcaaggacga cggcaactac
     1621 aagacccgcg ccgaggtgaa gttcgagggc gacaccctgg tgaaccgcat cgagctgaag
     1681 ggcatcgact tcaaggagga cggcaacatc ctggggcaca agctggagta caactacaac
     1741 agccacaacg tctatatcat ggccgacaag cagaagaacg gcatcaaggt gaacttcaag
     1801 atccgccaca acatcgagga cggcagcgtg cagctcgccg accactacca gcagaacacc
     1861 cccatcggcg acggccccgt gctgctgccc gacaaccact acctgagcac ccagtccgcc
     1921 ctgagcaaag accccaacga gaagcgcgat cacatggtcc tgctggagtt cgtgaccgcc
     1981 gccgggatca ctctcggcat ggacgagctg tacaaggagt tctaactaga gctcgctgat
     2041 cagcctcgaa acttgtttat tgcagcttat aatggttaca aataaagcaa tagcatcaca
     2101 aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc caaactcatc
     2161 aatgtatctt aaggtgggcc agaacatgtg ctgtctgcac agacaatgcg ctctgctctc
     2221 caaatggtga tgggcagtga ctgtaaaatc aagttagatg agtttgttct cttaatagca
     2281 gctacagttg ttgccctata atcttgagga agccgacccg cagtcctaaa gccaaagcac
     2341 agtcctgccc cctacccttg tttactgtag ttttccctac acacacagaa caatggtgaa
     2401 ttttggctta ttaattagac actataatat gtacaacaat aattaatagt acaaaagaac
     2461 aattataatg ctaatgaagt catatgagtg agttctatcc ttcctgttgt gatagtgaag
     2521 tgacacaaag tggtaagatg gaaggaagaa agtgggatag ttgttgtgat acacaaggct
     2581 gctgttgacc atctgtggct atgtcatcag aattttcaag tcatgcctct gccagctgac
     2641 ctcaaagaac taaaagtgca gctctcaatg gctttgggta tgctgtgtaa catttcagcg
     2701 tttggtgtga aggatccact agttctagag cggccgccac cgcggtggag ctccagcttt
     2761 tgttcccttt agtgagggtt aatttcgagc ttggcgtaat catggtcata gctgtttcct
     2821 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     2881 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     2941 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     3001 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     3061 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     3121 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     3181 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     3241 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     3301 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     3361 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     3421 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     3481 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     3541 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     3601 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     3661 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     3721 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     3781 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     3841 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     3901 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     3961 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     4021 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     4081 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     4141 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     4201 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     4261 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     4321 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     4381 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     4441 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     4501 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     4561 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     4621 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     4681 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     4741 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     4801 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     4861 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     4921 ggggttccgc gcacatttcc ccgaaaagtg ccac
//
