LOCUS       sequence-339946-        6851 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MegaGate destination with C-terminal DD tag and blasticidin 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6851)
  AUTHORS   Kramme C, Plesa AM, Wang HH, Wolf B, Smela MP, Guo X, Kohman RE, 
            Chatterjee P, Church GM
  TITLE     An integrated pipeline for mammalian genetic screening.
  JOURNAL   Cell Rep Methods. 2021 Sep 27;1(6):100082. doi: 
            10.1016/j.crmeth.2021.100082. eCollection 2021 Oct 25.
  PUBMED    35474898
REFERENCE   2  (bases 1 to 6851)
  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: "Cell Rep 
            Methods."; date: "2021-09-27"; volume: "1(6):100082. doi"; pages: " 
            10.1016/j.crmeth.2021.100082. eCollection 2021 Oct 25"
COMMENT     Sequence Label: pPB-EF1a-MegaGate-DD-Blast
FEATURES             Location/Qualifiers
     source          1..6851
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   418..480
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    752..783
                     /label=rox
                     /bound_moiety="Dre recombinase"
     polyA_signal    831..1055
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     1044..1061
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     CDS             complement(1083..1481)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     regulatory      1478..1487
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(1501..2000)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(1549..1568)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     1942..1965
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    2050..2081
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        2103..3285
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2334..3276
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3233..3253
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     3287..3311
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    3357..3481
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(3602..3726)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3728..4051
                     /codon_start=1
                     /product="destabilization domain that can be stabilized by 
                     Shield1 in the ProteoTuner(TM) system"
                     /label=DD-C
                     /note="FKBP12 mutant optimized for use as a C-terminal tag"
                     /translation="GVQVETISPGDGRTFPKRGQTCVVHYTGMLGDGKKVDSSRDRNKP
                     FKFMLGKQEVIRGWEEGVAQMSVGQGAKLTISPDYAYGATGHPGIIPPHATLVFDVELL
                     ELE"
     polyA_signal    4164..4298
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4242..4261
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4570..4604
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4630..4646)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4630..4646)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4643..4665)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4654..4670
                     /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(4678..4684,4685..4702,4703..4708))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4723..4744
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4861..4878)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5032..5620)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5112..5131)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5791..6582,6583..6651))
                     /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     6414..6433
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6652..6756)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6824..6842
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt caagctttat tatttaaccc
      421 tagaaagata atcatattgt gacgtacgtt aaagataatc atgcgtaaaa ttgacgcatg
      481 tgttttatcg gtctgtatat cgaggtttat ttattaattt gaatagatat taagttttat
      541 tatatttaca cttacatact aataataaat tcaacaaaca atttatttat gtttatttat
      601 ttattaaaaa aaaacaaaaa ctcaaaattt cttctataaa gtaacaaaac ttttaaacat
      661 tctctctttt acaaaaataa acttattttg tactttaaaa acagtcatgt tgtaatcgat
      721 aataagtaat tagcctagat atcggcgcgc ctaactttaa ataattggca ttatttaaag
      781 ttaagtactc tagtatgtcg agccccagct ggttctttcc gcctcagaag ccatagagcc
      841 caccgcatcc ccagcatgcc tgctattgtc ttcccaatcc tcccccttgc tgtcctgccc
      901 caccccaccc cccagaatag aatgacacct actcagacaa tgcgatgcaa tttcctcatt
      961 ttattaggaa aggacagtgg gagtggcacc ttccagggtc aaggaaggca cgggggaggg
     1021 gcaaacaaca gatggctggc aactagaagg cacagtcgag gctgatcagc gagctctaga
     1081 gcttagccct cccacacata accagagggc agcaattcac gaatcccaac tgccgtcggc
     1141 tgtccatcac tgtccttcac tatggctttg atcccaggat gcagatcgag aagcacctgt
     1201 cggcaccgtc cgcaggggct caagatgccc ctgttctcat ttccgatcgc gacgatacaa
     1261 gtcaggttgc cagctgccgc agcagcagca gtgcccagca ccacgagttc tgcacaaggt
     1321 cccccagtaa aatgatatac attgacacca gtgaagatgc ggccgtcgct agagagagct
     1381 gcgctggcga cgctgtagtc ttcagagatg gggatgctgt tgattgtagc cgttgctctt
     1441 tcaatgaggg tggattcttc ttgagacaaa ggcttggcca tggtggcgtt ggctgcaggt
     1501 cgaaaggccc ggagatgagg aagaggagaa cagcgcggca gacgtgcgct tttgaagcgt
     1561 gcagaatgcc gggcctccgg aggaccttcg ggcgcccgcc ccgcccctga gcccgcccct
     1621 gagcccgccc ccggacccac cccttcccag cctctgagcc cagaaagcga aggagcaaag
     1681 ctgctattgg ccgctgcccc aaaggcctac ccgcttccat tgctcagcgg tgctgtccat
     1741 ctgcacgaga ctagtgagac gtgctacttc catttgtcac gtcctgcacg acgcgagctg
     1801 cggggcgggg gggaacttcc tgactagggg aggagtagaa ggtggcgcga aggggccacc
     1861 aaagaacgga gccggttggc gcctaccggt ggatgtggaa tgtgtgcgag gccagaggcc
     1921 acttgtgtag cgccaagtgc ccagcggggc tgctaaagcg catgctccag actgccttgg
     1981 gaaaagcgcc tcccctaccc ggtagaatta attcgatatc aagctgaagt tcctatactt
     2041 tctcatatgt aactttaaat aattggcatt atttaaagtt attacttgta cagctcgtcc
     2101 atggctccgg tgcccgtcag tgggcagagc gcacatcgcc cacagtcccc gagaagttgg
     2161 ggggaggggt cggcaattga accggtgcct agagaaggtg gcgcggggta aactgggaaa
     2221 gtgatgtcgt gtactggctc cgcctttttc ccgagggtgg gggagaaccg tatataagtg
     2281 cagtagtcgc cgtgaacgtt ctttttcgca acgggtttgc cgccagaaca caggtaagtg
     2341 ccgtgtgtgg ttcccgcggg cctggcctct ttacgggtta tggcccttgc gtgccttgaa
     2401 ttacttccac gcccctggct gcagtacgtg attcttgatc ccgagcttcg ggttggaagt
     2461 gggtgggaga gttcgaggcc ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag
     2521 gcctggcttg ggcgctgggg ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc
     2581 gctgctttcg ataagtctct agccatttaa aatttttgat gacctgctgc gacgcttttt
     2641 ttctggcaag atagtcttgt aaatgcgggc caagatctgc acactggtat ttcggttttt
     2701 ggggccgcgg gcggcgacgg ggcccgtgcg tcccagcgca catgttcggc gaggcggggc
     2761 ctgcgagcgc ggccaccgag aatcggacgg gggtagtctc aagctggccg gcctgctctg
     2821 gtgcctggcc tcgcgccgcc gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg
     2881 gcaccagttg cgtgagcgga aagatggccg cttcccggcc ctgctgcagg gagctcaaaa
     2941 tggaggacgc ggcgctcggg agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc
     3001 tttccgtcct cagccgtcgc ttcatgtgac tccacggagt accgggcgcc gtccaggcac
     3061 ctcgattagt tctcgagctt ttggagtacg tcgtctttag gttgggggga ggggttttat
     3121 gcgatggagt ttccccacac tgagtgggtg gagactgaag ttaggccagc ttggcacttg
     3181 atgtaattct ccttggaatt tgcccttttt gagtttggat cttggttcat tctcaagcct
     3241 cagacagtgg ttcaaagttt ttttcttcca tttcaggtgt cgtgagagct cgtttagtga
     3301 accgtcagat cgcctggaga cgccatccac gctgttttga cctccataga agacacacaa
     3361 gtttgtacaa aaaagctgaa cgagaaacgt aaaatgatat aaatatcaat atattaaatt
     3421 agattttgca taaaaaacag actacataat actgtaaaac acaacatatc cagtcactat
     3481 ggcgacagaa gaagtatagg gataacaggg taattgttgt aagcgcgcta tgatggaggc
     3541 tatgccacta gaatctgcgt tcgctacctt aggaccgtta tagttagaag gaaagctcca
     3601 tcatagtgac tggatatgtt gtgttttaca gtattatgta gtctgttttt tatgcaaaat
     3661 ctaatttaat atattgatat ttatatcatt ttacgtttct cgttcagctt tcttgtacaa
     3721 agtggtagga gtgcaggtgg aaaccatctc cccaggagat gggcgcacct tccccaagcg
     3781 cggccagacc tgcgtggtgc actacaccgg gatgcttgga gatggaaaga aagttgactc
     3841 ctcccgggac agaaacaagc cctttaagtt tatgctaggc aagcaggagg tgatccgagg
     3901 ctgggaagaa ggggttgccc agatgagtgt gggtcaggga gccaaactga ctatatctcc
     3961 agattatgcc tatggtgcca ctgggcaccc aggcatcatc ccaccacatg ccactctcgt
     4021 gttcgatgtg gagcttctag aactggaatg agaaatactc agaagatgtc actcaccata
     4081 gagagaccat taatgggtct cctaggccac tgagcaagaa ctatgaatga gatctttaga
     4141 catattgcgg ccgctttctc cgcgatccag acatgataag atacattgat gagtttggac
     4201 aaaccaaaac tagaatgcag tgaaaaaaat gccttatttg tgaaatttgt gatgctattg
     4261 ccttatttgt aaccattata agctgcaata aacaagttat ctataacaag aaaatatata
     4321 tataataagt tatcacgtaa gtagaacatg aaataacaat ataattatcg tatgagttaa
     4381 atcttaaaag tcacgtaaaa gataatcatg cgtcattttg actcacgcgg tcgttatagt
     4441 tcaaaatcag tgacacttac cgcattgaca agcacgcctc acgggagctc caagcggcga
     4501 ctgagatgtc ctaaatgcac agcgacggat tcgcgctatt tagaaagaga gagcaatatt
     4561 tcaagaatgc atgcgtcaat tttacgcaga ctatctttct agggttaaaa aaagcttggc
     4621 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     4681 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     4741 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     4801 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgca cttccgcttc
     4861 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     4921 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     4981 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     5041 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     5101 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     5161 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     5221 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     5281 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct
     5341 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     5401 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     5461 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     5521 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     5581 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     5641 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     5701 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     5761 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     5821 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     5881 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gtgacccacg
     5941 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     6001 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     6061 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     6121 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     6181 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     6241 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     6301 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     6361 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     6421 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     6481 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     6541 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     6601 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     6661 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     6721 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc
     6781 tgacgtctaa gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag
     6841 gccctttcgt c
//
