LOCUS       Exported                8458 bp ds-DNA     circular SYN 24-MAY-2022
DEFINITION  Big-IN Landing Pad plasmid encoding pEF1a-driven 
            PuroR-P2A-hmPIGA-P2A-mScarlet expression cassette flanked by 
            heterotypic LoxM/LoxP sites and BsaI homology arms cloning sites..
ACCESSION   .
VERSION     .
KEYWORDS    pLP-PIGA2 (pLP300)
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8458)
  AUTHORS   Brosh R, Laurent JM, Ordonez R, Huang E, Hogan MS, Hitchcock AM, 
            Mitchell LA, Pinglay S, Cadley JA, Luther RD, Truong DM, Boeke JD, 
            Maurano MT
  TITLE     A versatile platform for locus-scale genome rewriting and 
            verification.
  JOURNAL   Proc Natl Acad Sci U S A. 2021 Mar 9;118(10). pii: 2023952118. doi: 
            10.1073/pnas.2023952118.
  PUBMED    33649239
REFERENCE   2  (bases 1 to 8458)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 24, 2022 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8458
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        complement(144..627)
                     /label=hPGK promoter
                     /note="human phosphoglycerate kinase 1 promoter"
     primer_bind     complement(180..198)
                     /label=hPGK-F
                     /note="Human PGK promoter, forward primer"
     protein_bind    680..713
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     promoter        720..1898
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          951..1889
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1846..1866
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     CDS             1905..2501
                     /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(1905..1924)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     2401..2421
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     CDS             2517..2573
                     /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"
     CDS             4044..4100
                     /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"
     CDS             4107..4802
                     /codon_start=1
                     /product="bright monomeric red fluorescent protein evolved 
                     from a synthetic template (Bindels et al., 2016)"
                     /label=mScarlet
                     /translation="MVSKGEAVIKEFMRFKVHMEGSMNGHEFEIEGEGEGRPYEGTQTA
                     KLKVTKGGPLPFSWDILSPQFMYGSRAFTKHPADIPDYYKQSFPEGFKWERVMNFEDGG
                     AVTVTQDTSLEDGTLIYKVKLRGTNFPPDGPVMQKKTMGWEASTERLYPEDGVLKGDIK
                     MALRLKDGGRYLADFKTTYKAKKPVQMPGAYNVDRKLDITSHNEDYTVVEQYERSEGRH
                     STGGMDELYK"
     primer_bind     complement(4240..4258)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     protein_bind    5218..5251
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GCATACAT)."
     primer_bind     complement(5362..5378)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5362..5378)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5390..5407)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5561..6149)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5641..5660)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6320..7180)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLRNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERKRQIAEIGAS
                     LIKHW"
     primer_bind     6943..6962
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7181..7285)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7353..7371
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     7380..7397
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     7381..7397
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     polyA_signal    7479..7600
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(7490..7509)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     7544..7563
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             complement(7601..138)
                     /codon_start=1
                     /product="truncated herpes simplex virus thymidine kinase 
                     (Salomon et al., 1995)"
                     /label=-Delta-TK
                     /note="confers sensitivity to nucleoside analogs such as 
                     acyclovir or ganciclovir"
                     /translation="MPTLLRVYIDGPHGMGKTTTTQLLVALGSRDDIVYVPEPMTYWRV
                     LGASETIANIYTTQHRLDQGEISAGDAAVVMTSAQITMGMPYAVTDAVLAPHIGGEAGS
                     SHAPPPALTLIFDRHPIAALLCYPAARYLMGSMTPQAVLAFVALIPPTLPGTNIVLGAL
                     PEDRHIDRLAKRQRPGERLDLAMLAAIRRVYGLLANTVRYLQCGGSWREDWGQLSGTAV
                     PPQGAEPQSNAGPRPHIGDTLFTLFRAPELLAPNGDLYNVFAWALDVLAKRLRSMHVFI
                     LDYDQSPAGCRDALLQLTSGMVQTHVTTPGSIPTICDLARTFAREMGEAN"
ORIGIN
        1 cagcacccgc cagtaagtca tcggctcggg tacgtagacg atatcgtcgc gcgaacccag
       61 ggccaccagc agttgcgtgg tggtggtttt ccccatcccg tggggaccgt ctatataaac
      121 ccgcagtagc gtgggcatcc tccaacgagg gagccgactg ccgacgtgcg ctccggaggc
      181 ttgcagaatg cggaacaccg cgcgggcagg aacagggccc acactaccgc cccacacccc
      241 gcctcccgca ccgccccttc ccagccgctg ctctcggcgc gccctgctga gcagccgcta
      301 ttggccacag cccatcgcgg tcggcgcgct gccattgctc cctggcgctg tccgtctgcg
      361 agggtactag tcagacgtgc ggcttccgtt tgtcacgtcc ggcacgccgc gaaccgcaag
      421 gaaccttccc gacttagggg cggagcagga agcgtcgccg gggggcccac aagggtagcg
      481 gcgaagatcc gggtgacgct gcgaacggac gtgaagaatg tgcgaggccc agggtcggcg
      541 ccgctgcgtt tcccggaacc acgcccagag cagccgcgtc cctgcgcaaa cccagggctg
      601 ccttggaaaa ggcgcaaccc caacccccct gcaggcgtct ctgagcgccc tcgagacctt
      661 atacgcacgg tctcaaacga taacttcgta taggatactt tatacgaagt tataccggtg
      721 gctccggtgc ccgtcagtgg gcagagcgca catcgcccac agtccccgag aagttggggg
      781 gaggggtcgg caattgaagc ggtgcctaga gaaggtggcg cggggtaaac tgggaaagtg
      841 atgtcgtgta ctggctccgc ctttttcccg agggtggggg agaaccgtat ataagtgcag
      901 tagtcgccgt gaacgttctt tttcgcaacg ggtttgccgc cagaacacag gtaagtgccg
      961 tgtgtggttc ccgcgggcct ggcctcttta cgggttatgg cccttgcgtg ccttgaatta
     1021 cttccacctg gctgcagtac gtgattcttg atcccgagct tcgggttgga agtgggtggg
     1081 agagttcgag gccttgcgct taaggagccc cttcgcctcg tgcttgagtt gaggcctggc
     1141 ctgggcgctg gggccgccgc gtgcgaatct ggtggcacct tcgcgcctgt ctcgctgctt
     1201 tcgataagtc tctagccatt taaaattttt gatgacctgc tgcgacgctt tttttctggc
     1261 aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt tttggggccg
     1321 cgggcggcga cggggcccgt gcgtcccagc gcacatgttc ggcgaggcgg ggcctgcgag
     1381 cgcggccacc gagaatcgga cgggggtagt ctcaagctgg ccggcctgct ctggtgccta
     1441 gtctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg tcggcaccag
     1501 ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca aaatggagga
     1561 cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg gcctttccgt
     1621 cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg cacctcgatt
     1681 agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt tatgcgatgg
     1741 agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac ttgatgtaat
     1801 tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag cctcagacag
     1861 tggttcaaag tttttttctt ccatttcagg tgtcgtgacg gccgatgacc gagtacaagc
     1921 ccacggtgcg cctcgccacc cgcgacgacg tccccagggc cgtacgcacc ctcgccgccg
     1981 cgttcgccga ctaccccgcc acgcgccaca ccgtcgatcc ggaccgccac atcgagcggg
     2041 tcaccgagct gcaagaactc ttcctcacgc gcgtcgggct cgacatcggc aaggtgtggg
     2101 tcgcggacga cggcgccgcg gtggcggtct ggaccacgcc ggagagcgtc gaagcggggg
     2161 cggtgttcgc cgagatcggc ccgcgcatgg ccgagttgag cggttcccgg ctggccgcgc
     2221 agcaacagat ggaaggcctc ctggcgccgc accggcccaa ggagcccgcg tggttcctgg
     2281 ccaccgtcgg cgtgtcgccc gaccaccagg gcaagggtct gggcagcgcc gtcgtgctcc
     2341 ccggagtgga ggccgccgag cgcgccgggg tgcccgcctt cctggaaacc tccgcgcccc
     2401 gcaacctccc cttctacgag cggctcggct tcaccgtcac cgccgacgtc gaggtgcccg
     2461 aaggaccgcg cacctggtgc atgacccgca agcccggtgc cgtcgacggc agcggcgcca
     2521 ccaacttcag cctgctgaag caggccggcg acgtggagga gaaccccggg cccgaattcg
     2581 cctgtagagg aggagctggg aatggccacc gtgcctcagc tacactctct cgggttagcc
     2641 ctggaagtct ttacacatgt agaacccgta cccataatat ctgcatggta tctgactttt
     2701 tctacccaaa tatgggaggc gtggaaagcc acatttacca gctctctcag tgcctgattg
     2761 aaagagggca taaggttata attgtcaccc atgcttatgg aaatcgaaaa ggcatccgtt
     2821 acctcaccag tggcctcaaa gtctattact tgcctctgaa agtcatgtac aaccagtcta
     2881 cagccacgac cctctttcac agtctgccat tgctcaggta catatttgtt cgggagagag
     2941 tcacgataat ccattcacat agttcttttt ctgctatggc ccatgatgct ctcttccacg
     3001 ccaagacaat ggggcttcag acagtgttca cggaccattc cctttttgga tttgctgatg
     3061 tcagctcggt gcttacaaac aagctgctaa ccgtgtctct ttgtgataca aaccacatca
     3121 tttgtgtgtc ttatactagt aaggaaaata ctgtactaag agcagcactg aatcctgaaa
     3181 tagtgtccgt cattcctaat gctgtagatc ctactgactt cactccagac ccatttagaa
     3241 ggcatgatag tataactatt gttgttgtca gcagacttgt ttacagaaaa gggatcgatt
     3301 tgcttagtgg tataatacct gaactctgtc agaaatatcc agatttaaat ttcataattg
     3361 gaggagaggg accaaagaga atcattttgg aagaagttcg ggaaagatac cagctgcatg
     3421 acagggtgcg tcttttggga gctttagaac acaaggatgt tagaaatgtc ttagttcaag
     3481 gacatatttt tctgaatacc tcccttactg aagcattctg catggccatc gtggaagcag
     3541 ccagttgtgg tttacaggtt gtaagtacca gagttggtgg gattcctgag gtgcttccag
     3601 aaaaccttat tattttatgt gagccttcag taaaatcttt gtgtgaagga ttggaaaagg
     3661 ctattttcca actgaagtca gggacattgc cagctccaga aaacatccat aacatagtaa
     3721 agactttcta cacctggagg aatgttgcag aaagaactga aaaggtatat gaccgggtat
     3781 cagtggaagc tgtgttgcca atggacaaac gactggacag acttatttct cactgcggcc
     3841 cagtaacagg ctacatcttt gctttgttgg cagttttcaa cttcctcttc ctcattttct
     3901 tgagatggat gactccagat tctatcattg atgttgcaat agatgccact gggccacggg
     3961 gtgcctggac taataactat tctcacagta aaagaggggg tgagaataat gagatatctg
     4021 aaaccagggg atccggctca ggcgccacca actttagcct gctgaagcag gcaggcgacg
     4081 tcgaggagaa ccccggccct acgcgtatgg tgagcaaggg cgaggcagtg atcaaggagt
     4141 tcatgcggtt caaggtgcac atggagggct ccatgaacgg ccacgagttc gagatcgagg
     4201 gcgagggcga gggccgcccc tacgagggca cccagaccgc caagctgaag gtgaccaagg
     4261 gtggccccct gcccttctcc tgggacatcc tgtcccctca gttcatgtac ggctccaggg
     4321 ccttcaccaa gcaccccgcc gacatccccg actactataa gcagtccttc cccgagggct
     4381 tcaagtggga gcgcgtgatg aacttcgagg acggcggcgc cgtgaccgtg acccaggaca
     4441 cctccctgga ggacggcacc ctgatctaca aggtgaagct ccgcggcacc aacttccctc
     4501 ctgacggccc cgtaatgcag aagaaaacaa tgggctggga agcgtccacc gagcggttgt
     4561 accccgagga cggcgtgctg aagggcgaca ttaagatggc cctgcgcctg aaggacggag
     4621 gccgctacct ggcggacttc aagaccacct acaaggccaa gaagcccgtg cagatgcccg
     4681 gcgcctacaa cgttgaccgc aagttggaca tcacctccca caacgaggac tacaccgtgg
     4741 tggaacagta cgaacgctcc gagggccgcc actccaccgg cggcatggac gagctgtaca
     4801 agtaaggtac ctgaggccaa ccttctatca gagttaaact tttgacaagg gaacaaatct
     4861 caaactgatc catcagtcat gtagctagct gtagagcttg caacttaata gcagcagctg
     4921 cccaatgcca tgtgaagtaa caaactggtt tttggttttt ttttcccctt cagttttaat
     4981 gttatgtgta atgtatttaa acccttattt aaataaaact tgttttcaga aatacctgac
     5041 ttgcagatta tttatatata gtcgatcttg tgcatataaa ggaaccatag aaatgggcag
     5101 tggaggattc gtccacaccc acagcttctc aacgggcctg aatttggcca agaaaaacct
     5161 ttatgagctt gtccctctct ggtcaactgg ttttttgttc gtttgttttt tgagacaata
     5221 acttcgtata gcatacatta tacgaagtta ttatgcgaga ccttatacgc acggtctcaa
     5281 tccgttcaag agacgatttc gggtccacaa cgaccttgaa aaactatttc gggagagacg
     5341 gagtcactgc caaccgagac ggtcatagct gtttcctgtg tgccgcttcc tcgctcactg
     5401 actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa
     5461 tacggttacc cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc
     5521 aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc
     5581 ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat
     5641 aaagatacca ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc
     5701 cgcttaccgg atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct
     5761 cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg
     5821 aaccccccgt tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc
     5881 cggtaagaca cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga
     5941 ggtatgtagg cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa
     6001 gaacagtatt tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta
     6061 gctcttgatc cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc
     6121 agattacgcg cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg
     6181 acgctcagtg gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga
     6241 tcttcaccta gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg
     6301 agtaaacttg gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct
     6361 gtctctttcg ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg
     6421 agggcttacc atctggcccc agtgctgcaa taataccgcg ggacccacgc tcaccggctc
     6481 cagatttatc agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa
     6541 ctttatccgc ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc
     6601 cagttaatag tttgcgcaac gttgttgcca tcgctacagg catcgtggta tcacgctcgt
     6661 cgtttggtat ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc
     6721 ccatgttgcg caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt
     6781 tggccgccgt gttatcactc atggttatgg cagcactaca taattctctt actgtcatgc
     6841 catccgtaag atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt
     6901 gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata
     6961 gcagaacttt aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga
     7021 tcttaccgct gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag
     7081 catcttttac tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa
     7141 aaaagggaat aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt
     7201 attgaagcat ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga
     7261 aaaataaaca aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag
     7321 aaaccattat tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtt
     7381 gtaaaacgac ggccagtcga accacgcaat gcgtctcgat ccgcagtgtc ttgcgtctct
     7441 ggcaaaatca tgtataaggt gaagggcatc cctgcaggta agatacattg atgagtttgg
     7501 acaaaccaca actagaatgc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     7561 tgctttattt gtaaccatta taagctgcaa taaacaagtt ttagttagcc tcccccatct
     7621 cccgggcaaa cgtgcgcgcc aggtcgcata tcgtcggtat ggagccgggg gtggtgacgt
     7681 gggtctggac catcccggag gtaagttgca gcagggcgtc ccggcagccg gcgggcgatt
     7741 ggtcgtaatc caggataaag acgtgcatgg aacggaggcg tttggccaag acgtccaagg
     7801 cccaggcaaa cacgttatac aggtcgccgt tgggggccag caactcgggg gcccgaaaca
     7861 gggtaaataa cgtgtccccg atatggggtc gtgggcccgc gttgctctgg ggctcggcac
     7921 cctggggcgg cacggcggtc cccgaaagct gtccccagtc ctcccgccac gacccgccgc
     7981 attgcagata ccgcaccgta ttggcaagta gcccgtaaac gcggcgaatc gcagccagca
     8041 tagccaggtc cagccgctcg ccggggcgct ggcgtttggc caggcggtcg atgtgtctgt
     8101 cctccggaag ggccccaagc acgatgttgg tgccgggcaa ggtcggcggg atgagggcca
     8161 cgaacgccag cacggcctgg ggggtcatgc tgcccataag gtagcgcgcg gcggggtagc
     8221 acaggagggc ggcgatggga tggcggtcga agatgagggt gagggccggg ggcggggcat
     8281 gtgagctccc agcctccccc ccgatatgag gagccagaac ggcgtcggtc acggcataag
     8341 gcatccccat tgttatctgg gcgcttgtca ttaccaccgc cgcgtccccg gcggatatct
     8401 caccctggtc gaggcggtgt tgtgtggtgt agatgttcgc gattgtctcg gaagcccc
//
