LOCUS       sequence-389805-        6287 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  Big-IN Landing Pad plasmid encoding pEF1a-driven PuroR-P2A-hmPIGA 
            expression cassette flanked by heterotypic LoxM/LoxP sites, sleeping
            beauty ITRs and BsmBI homology arms cloning sites. .
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6287)
  AUTHORS   Brosh R, Coelho C, Ribeiro-Dos-Santos AM, Ellis G, Hogan MS, Ashe 
            HJ, Somogyi N, Ordonez R, Luther RD, Huang E, Boeke JD, Maurano MT
  TITLE     Synthetic regulatory genomics uncovers enhancer context dependence 
            at the Sox2 locus.
  JOURNAL   Mol Cell. 2023 Apr 6;83(7):1140-1152.e7. doi: 
            10.1016/j.molcel.2023.02.027. Epub 2023 Mar 16.
  PUBMED    36931273
REFERENCE   2  (bases 1 to 6287)
  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.molcel.2023.02.027"; journalName: "Mol Cell"; date: 
            "2023-04-6- 6"; volume: "83"; issue: "7"; pages: "1140-1152.e7"
COMMENT     Sequence Label: pLP-PIGA3 (pLP305)
FEATURES             Location/Qualifiers
     source          1..6287
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    complement(240..273)
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (AAGTATCC) (Shaw et al., 2021). lox2272 sites are 
                     compatible with each other, but incompatible with loxP or 
                     loxN sites (Lee and Saito, 1988)."
     promoter        300..1478
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          531..1469
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1426..1446
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1493..2089
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(1493..1512)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     1989..2009
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2099..2155
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     protein_bind    complement(4084..4117)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             complement(join(4492..5283,5284..5352))
                     /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     5115..5134
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(5353..5457)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      5560..6148
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     6049..6068
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 aacgtacagt tgaagtcgga agtttacata cacttaagtt ggagtcatta aaactcgttt
       61 ttcaactact ccacaaattt cttgttaaca aacaatagtt ttggcaagtc agttaggaca
      121 tctactttgt gcatgacaca agtcattttt ccaacaattg tttacagaca gattatttca
      181 cttataattc actgtatcac aattccagtg ggtcagaagt ttacatacac taagtaacga
      241 taacttcgta taggatactt tatacgaagt tatacgcgcc tgcagcagac gtaagtcggg
      301 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
      361 gaggggtcgg caattgaacc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
      421 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
      481 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
      541 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
      601 cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg
      661 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc
      721 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt
      781 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc
      841 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg
      901 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag
      961 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgccta
     1021 gtctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag
     1081 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga
     1141 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt
     1201 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gctgtccagg cacctcgatt
     1261 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg
     1321 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat
     1381 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag
     1441 tggttcaaag tttttttctt ccatttcagg tgtcgtgaga gcccatgtcg acatgaccga
     1501 gtacaagccc acggtgcgcc tcgccacccg cgacgacgtc cccagggccg tacgcaccct
     1561 cgccgccgcg ttcgccgact accccgccac gcgccacacc gtcgatccgg accgccacat
     1621 cgagcgggtc accgagctgc aagaactctt cctcacgcgc gtcgggctcg acatcggcaa
     1681 ggtgtgggtc gcggacgacg gcgccgcggt ggcggtctgg accacgccgg agagcgtcga
     1741 agcgggggcg gtgttcgccg agatcggccc gcgcatggcc gagttgagcg gttcccggct
     1801 ggccgcgcag caacagatgg aaggcctcct ggcgccgcac cggcccaagg agcccgcgtg
     1861 gttcctggcc accgtcggcg tgtcgcccga ccaccagggc aagggtctgg gcagcgccgt
     1921 cgtgctcccc ggagtggagg cggccgagcg cgccggggtg cccgccttcc tggaaacctc
     1981 cgcgccccgc aacctcccct tctacgagcg gctcggcttc accgtcaccg ccgacgtcga
     2041 ggtgcccgaa ggaccgcgca cctggtgcat gacccgcaag cccggtgccg gcagcggcgc
     2101 caccaacttc agcctgctga agcaggccgg cgacgtggag gagaaccccg ggcccgcctg
     2161 tagaggagga gctgggaatg gccaccgtgc ctcagctaca ctctctcggg ttagccctgg
     2221 aagtctttac acatgtagaa cccgtaccca taatatatgc atggtatctg actttttcta
     2281 cccaaatatg ggaggcgtgg aaagccacat ttaccagctc tctcagtgcc tgattgaaag
     2341 agggcataag gttataattg tcacccatgc ttatggaaat cgaaaaggca tccgttacct
     2401 caccagtggc ctcaaagtct attacttgcc tctgaaagtc atgtacaacc agtctacagc
     2461 cacgaccctc tttcacagtc tgccattgct caggtacata tttgttcggg agagagtcac
     2521 gataatccat tcacatagtt ctttttctgc tatggcccat gatgctctct tccacgccaa
     2581 gacaatgggg cttcagacag tgttcacgga ccattccctt tttggatttg ctgatgtcag
     2641 ctcggtgctt acaaacaagc ttctaaccgt gtctctttgt gatacaaacc acatcatttg
     2701 tgtgtcttat actagtaagg aaaatactgt actaagagca gcactgaatc ctgaaatagt
     2761 gtccgtcatt cctaatgctg tagatcctac tgacttcact ccagacccat ttagaaggca
     2821 tgatagtata actattgttg ttgtcagcag acttgtttac agaaaaggga tcgatttgct
     2881 tagtggtata atacctgaac tctgtcagaa atatccagat ttaaatttca taattggagg
     2941 agagggacca aagagaatca ttttggaaga agttcgggaa agataccagc tgcatgacag
     3001 ggtgcgtctt ttgggagctt tagaacacaa ggatgttaga aatgtcttag ttcaaggaca
     3061 tatttttctg aatacctccc ttactgaagc attctgcatg gcgatcgtgg aagcagccag
     3121 ttgtggttta caggttgtaa gtaccagagt tggtgggatt cctgaggtgc ttccagaaaa
     3181 ccttattatt ttatgtgagc cttcagtaaa atctttgtgt gaaggattgg aaaaggctat
     3241 tttccaactg aagtcaggga cattgccagc tccagaaaac atccataaca tagtaaagac
     3301 tttctacacc tggaggaatg ttgcagaaag aactgaaaag gtatatgacc gggtatcagt
     3361 ggaagctgtg ttgccaatgg acaaacgact ggacagactt atttctcact gcggcccagt
     3421 aacaggctac atctttgctt tgttggcagt tttcaacttc ctcttcctca ttttcttgag
     3481 atggatgact ccagattcta tcattgatgt tgcaatagat gccactgggc cacggggtgc
     3541 ctggactaat aactattctc acagtaaaag agggggtgag aataatgaga tatctgaaac
     3601 caggtaaggt acctgaggcc aaccttctat cagagttaaa cttttgacaa gggaacaaat
     3661 ctcaaactga tccatcagtc atgtagctag ctgtagagct tgcaacttaa tagcagcagc
     3721 tgcccaatgc catgtgaagt aacaaactgg tttttggttt ttttttcccc ttcagtttta
     3781 atgttatgtg taatgtattt aaacccttat ttaaataaaa cttgttttca gaaatacctg
     3841 acttgcagat tatttatata tagtcgatct tgtgcatata aaggaaccat agaaatgggc
     3901 agtggaggat tcgtccacac ccacagcttc tcaacgggcc tgaatttggc caagaaaaac
     3961 ctttatgagc ttgtccctct ctggtcaact ggttttttgt tcgtttgttt tttgagacat
     4021 tcattgcaat agtgtgttgg aattttttgt gtctctcact cggtgcgtcc tgcaggcgcg
     4081 ttcataactt cgtatagcat acattatacg aagttattat gattgagtgt atgtaaactt
     4141 ctgacccact gggaatgtga tgaaagaaat aaaagctgaa atgaatcatt ctctctacta
     4201 ttattctgat atttcacatt cttaaaataa agtggtcatc ctaactgacc taagacaggg
     4261 aatttttact aggattaaat gtcaggaatt gtgaaaaagt gagtttaaat gtatttggct
     4321 aaggtgtatg taaacttccg acttcaactg tattatgcga gacgttatac gcaccgtctc
     4381 aatccatccg cggccgcgat tatcaaaaag gatcttcacc tagatccttt taaattaaaa
     4441 atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca gttaccaatg
     4501 cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca tagttgcctg
     4561 actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc ccagtgctgc
     4621 aatgataccg cgggacccac gctcaccggc tccagattta tcagcaataa accagccagc
     4681 cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc agtctattaa
     4741 ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca acgttgttgc
     4801 cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat tcagctccgg
     4861 ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag cggttagctc
     4921 cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac tcatggttat
     4981 ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt ctgtgactgg
     5041 tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt gctcttgccc
     5101 ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc tcatcattgg
     5161 aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat ccagttcgat
     5221 gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca gcgtttctgg
     5281 gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga cacggaaatg
     5341 ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg gttattgtct
     5401 catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg ttccgcgcac
     5461 atttccccga aaagtgccac ctgtcatgac caaaatccct taacgtgagt tttcgttcca
     5521 ctgagcgtca gaccccgtag aaaagatcaa aggatcttct tgagatcctt tttttctgcg
     5581 cgtaatctgc tgcttgcaaa caaaaaaacc accgctacca gcggtggttt gtttgccgga
     5641 tcaagagcta ccaactcttt ttccgaaggt aactggcttc agcagagcgc agataccaaa
     5701 tactgttctt ctagtgtagc cgtagttagg ccaccacttc aagaactctg tagcaccgcc
     5761 tacatacctc gctctgctaa tcctgttacc agtggctgct gccagtggcg ataagtcgtg
     5821 tcttaccggg ttggactcaa gacgatagtt accggataag gcgcagcggt cgggctgaac
     5881 ggggggttcg tgcacacagc ccagcttgga gcgaacgacc tacaccgaac tgagatacct
     5941 acagcgtgag ctatgagaaa gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc
     6001 ggtaagcggc agggtcggaa caggagagcg cacgagggag cttccagggg gaaacgcctg
     6061 gtatctttat agtcctgtcg ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg
     6121 ctcgtcaggg gggcggagcc tatggaaaaa cgccagcaac gcggcctttt tacggttcct
     6181 ggccttttgc tggccttttg ctcacatgtt ctttcctgcg ttatcccctg attctgtgga
     6241 taaccgtgcg gccgcccctc cctcgagacg ttatacgcac cgtctca
//
