LOCUS       sequence-466438-        4964 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Donor plasmid for knock-in of T2A-eGFP-SV40pA at the mouse Podxl 
            locus. Used with PX459-sgRNA047_Podxl sgRNA..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4964)
  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 4964)
  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: KL158 - pBlueScript II SK-T2A-eGFP-SV40pA Podx...
FEATURES             Location/Qualifiers
     source          1..4964
                     /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"
     CDS             1015..1068
                     /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(1069..1071,1072..1782)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="VSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITLGMDELYK"
     CDS             join(1069..1071,1072..1782)
                     /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(1111..1132)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(1372..1391)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1719..1740
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     polyA_signal    1816..1937
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1853..1872)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1907..1926
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     promoter        complement(2776..2794)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2815..2831)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2815..2831)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2828..2850)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2839..2855
                     /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(2863..2869,2870..2887,2888..2893))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2908..2929
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(3046..3063)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(3217..3805)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3297..3316)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3976..4767,4768..4836))
                     /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     4599..4618
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4837..4941)
                     /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 gtcgacagtc ttcgcagcaa cgtgcgcggg gccgcagcgg cgcagacacc
      721 gcccgggccg cagacacctt cgctcgtcgc tgctggctcc gtgtcccagc ccagctctgc
      781 ggtaggcagg aggagcggcc ggatcgcgac tccggagttc gcggccgtcg ccaggcctcc
      841 cggagcggcg ccgacgctcc cgctgtccgg acgcgcgacc ctgcgccagc gccgcagcca
      901 cctgctccga gtccccgggc gagcacaacc atgcctccca ctacggcgct ctccgcgctg
      961 ctgctgctgc tactgtcgcc tgcatctcac tcccataatg aattcggcag tggagagggc
     1021 agaggaagtc tgctaacatg cggtgacgtc gaggagaatc ctggcccagt gagcaagggc
     1081 gaggagctgt tcaccggggt ggtgcccatc ctggtcgagc tggacggcga cgtaaacggc
     1141 cacaagttca gcgtgtccgg cgagggcgag ggcgatgcca cctacggcaa gctgaccctg
     1201 aagttcatct gcaccaccgg caagctgccc gtgccctggc ccaccctcgt gaccaccctg
     1261 acctacggcg tgcagtgctt cagccgctac cccgaccaca tgaagcagca cgacttcttc
     1321 aagtccgcca tgcccgaagg ctacgtccag gagcgcacca tcttcttcaa ggacgacggc
     1381 aactacaaga cccgcgccga ggtgaagttc gagggcgaca ccctggtgaa ccgcatcgag
     1441 ctgaagggca tcgacttcaa ggaggacggc aacatcctgg ggcacaagct ggagtacaac
     1501 tacaacagcc acaacgtcta tatcatggcc gacaagcaga agaacggcat caaggtgaac
     1561 ttcaagatcc gccacaacat cgaggacggc agcgtgcagc tcgccgacca ctaccagcag
     1621 aacaccccca tcggcgacgg ccccgtgctg ctgcccgaca accactacct gagcacccag
     1681 tccgccctga gcaaagaccc caacgagaag cgcgatcaca tggtcctgct ggagttcgtg
     1741 accgccgccg ggatcactct cggcatggac gagctgtaca aggagttcta actagagctc
     1801 gctgatcagc ctcgaaactt gtttattgca gcttataatg gttacaaata aagcaatagc
     1861 atcacaaatt tcacaaataa agcatttttt tcactgcatt ctagttgtgg tttgtccaaa
     1921 ctcatcaatg tatcttaggt gagtggacag ccgcggctct gccgggaccc ctggtcattc
     1981 ccctccgccc caagcctggt ggggctcctg ggaagccggc tgcaccgcgt ggtcggaccg
     2041 cttgcagcct ctcccgcgtg gccgcacgca gagtcctgac cttggcacca tccacgcccc
     2101 ctccaaattg gagaaccccg caggtgcggg gccagatcgt ggatttattc ctagtcttca
     2161 ctggtgtaga gtgagccgga ggttgtgaga ttgtaaaggg ggcacttggt gggttcgctg
     2221 gagatggggg ggaaactttg ggttgtgggg aggtcccaga ttcccgcatg accaccagaa
     2281 tgcgatttct ctttctcgtg tttggggcta ctaggggacc gtattgggag gtcgttgaaa
     2341 ctaagcaaag ccgcgggcag ggaccacaag aaggagcccc tgaaactgac ctcaaatccc
     2401 taaatgccca cgcgtgtagc gtacttcttg tgcaagcgct ttttcttaag cttgtcccgg
     2461 gacgggtgga ataagcgctc cttccagggt cccagagcct ggttcagtgg ggtccttgcc
     2521 ttcatgttgc caaaggaccg gattctttct cagagcatgg ccaggatggt gggctcctcc
     2581 cacccctttg tgtaaacaag aaatttcttt cacaaatgag gtttgagttg tgtcttagta
     2641 atacctcggg cacccatctc ccatcagacc cagcttggag acgatcccac taccttcttt
     2701 ctgaaggtgg aatgttactt tctgtttgag aggtcaggcg cggccgccac cgcggtggag
     2761 ctccagcttt tgttcccttt agtgagggtt aatttcgagc ttggcgtaat catggtcata
     2821 gctgtttcct gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag
     2881 cataaagtgt aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg
     2941 ctcactgccc gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca
     3001 acgcgcgggg agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc
     3061 gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
     3121 gttatccaca gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
     3181 ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga
     3241 cgagcatcac aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag
     3301 ataccaggcg tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct
     3361 taccggatac ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg
     3421 ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
     3481 ccccgttcag cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt
     3541 aagacacgac ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta
     3601 tgtaggcggt gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac
     3661 agtatttggt atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc
     3721 ttgatccggc aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat
     3781 tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
     3841 tcagtggaac gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt
     3901 cacctagatc cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta
     3961 aacttggtct gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct
     4021 atttcgttca tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg
     4081 cttaccatct ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga
     4141 tttatcagca ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
     4201 atccgcctcc atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt
     4261 taatagtttg cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt
     4321 tggtatggct tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat
     4381 gttgtgcaaa aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc
     4441 cgcagtgtta tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc
     4501 cgtaagatgc ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat
     4561 gcggcgaccg agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag
     4621 aactttaaaa gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt
     4681 accgctgttg agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc
     4741 ttttactttc accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa
     4801 gggaataagg gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg
     4861 aagcatttat cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa
     4921 taaacaaata ggggttccgc gcacatttcc ccgaaaagtg ccac
//
