LOCUS       sequence-343088-        7478 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MegaGate destination with C-terminal DD tag and hygromycin 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7478)
  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 7478)
  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-Hygro
FEATURES             Location/Qualifiers
     source          1..7478
                     /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..2108)
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="MKKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRG
                     YVLRVNSCADGFYKDRYVHRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLP
                     ETELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQ
                     TVMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMF
                     GDSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDG
                     NFDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAK
                     E"
     promoter        complement(2128..2627)
                     /label=PGK promoter
                     /note="mouse phosphoglycerate kinase 1 promoter"
     primer_bind     complement(2176..2195)
                     /label=mPGK-F
                     /note="Mouse PGK promoter, forward primer"
     primer_bind     2569..2592
                     /label=MSCV-rev
                     /note="Murine stem cell virus, reverse primer"
     protein_bind    2677..2708
                     /label=rox
                     /bound_moiety="Dre recombinase"
     promoter        2730..3912
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          2961..3903
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     3860..3880
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     3914..3938
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    3984..4108
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(4229..4353)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             4355..4678
                     /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    4791..4925
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4869..4888
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   5197..5231
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(5257..5273)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5257..5273)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5270..5292)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5281..5297
                     /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(5305..5311,5312..5329,5330..5335))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5350..5371
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5488..5505)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5659..6247)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5739..5758)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6418..7209,7210..7278))
                     /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     7041..7060
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7279..7383)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7451..7469
                     /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 gcctattcct ttgccctcgg acgagtgctg gggcgtcggt ttccactatc ggcgagtact
     1141 tctacacagc catcggtcca gacggccgcg cttctgcggg cgatttgtgt acgcccgaca
     1201 gtcccggctc cggatcggac gattgcgtcg catcgaccct gcgcccaagc tgcatcatcg
     1261 aaattgccgt caaccaagct ctgatagagt tggtcaagac caatgcggag catatacgcc
     1321 cggagccgcg gcgatcctgc aagctccgga tgcctccgct cgaagtagcg cgtctgctgc
     1381 tccatacaag ccaaccacgg cctccagaag aagatgttgg cgacctcgta ttgggaatcc
     1441 ccgaacatcg cctcgctcca gtcaatgacc gctgttatgc ggccattgtc cgtcaggaca
     1501 ttgttggagc cgaaatccgc gtgcacgagg tgccggactt cggggcagtc ctcggcccaa
     1561 agcatcagct catcgagagc ctgcgcgacg gacgcactga cggtgtcgtc catcacagtt
     1621 tgccagtgat acacatgggg atcagcaatc gcgcatatga aatcacgcca cgtagtgtat
     1681 tgaccgattc cttgcggtcc gaatgggccg aacccgctcg tctggctaag atcggccgca
     1741 gcgatcgcat ccatggcctc cgcgaccggc tgcagaacag cgggcagttc ggtttcaggc
     1801 aggtcttgca acgtgacacc ctgtgcacgg cgggagatgc aataggtcag gctctcgctg
     1861 aattccccaa tgtcaagcac ttccggaatc gggagcgcgg ccgatgcaaa gtgccgatga
     1921 acataacgat ctttgtagaa accatcggcg cagctattta cccgcaggac atatccacgc
     1981 cctcctacat cgaagctgaa agcacgagat tcttcgccct ccgagagctg catcaggtcg
     2041 gagacgctgt cgaacttttc gatcagaaac ttctcgacag acgtcgcggt gagttcaggc
     2101 tttttcatgg tggcgttggc tgcaggtcga aaggcccgga gatgaggaag aggagaacag
     2161 cgcggcagac gtgcgctttt gaagcgtgca gaatgccggg cctccggagg accttcgggc
     2221 gcccgccccg cccctgagcc cgcccctgag cccgcccccg gacccacccc ttcccagcct
     2281 ctgagcccag aaagcgaagg agcaaagctg ctattggccg ctgccccaaa ggcctacccg
     2341 cttccattgc tcagcggtgc tgtccatctg cacgagacta gtgagacgtg ctacttccat
     2401 ttgtcacgtc ctgcacgacg cgagctgcgg ggcggggggg aacttcctga ctaggggagg
     2461 agtagaaggt ggcgcgaagg ggccaccaaa gaacggagcc ggttggcgcc taccggtgga
     2521 tgtggaatgt gtgcgaggcc agaggccact tgtgtagcgc caagtgccca gcggggctgc
     2581 taaagcgcat gctccagact gccttgggaa aagcgcctcc cctacccggt agaattaatt
     2641 cgatatcaag ctgaagttcc tatactttct catatgtaac tttaaataat tggcattatt
     2701 taaagttatt acttgtacag ctcgtccatg gctccggtgc ccgtcagtgg gcagagcgca
     2761 catcgcccac agtccccgag aagttggggg gaggggtcgg caattgaacc ggtgcctaga
     2821 gaaggtggcg cggggtaaac tgggaaagtg atgtcgtgta ctggctccgc ctttttcccg
     2881 agggtggggg agaaccgtat ataagtgcag tagtcgccgt gaacgttctt tttcgcaacg
     2941 ggtttgccgc cagaacacag gtaagtgccg tgtgtggttc ccgcgggcct ggcctcttta
     3001 cgggttatgg cccttgcgtg ccttgaatta cttccacgcc cctggctgca gtacgtgatt
     3061 cttgatcccg agcttcgggt tggaagtggg tgggagagtt cgaggccttg cgcttaagga
     3121 gccccttcgc ctcgtgcttg agttgaggcc tggcttgggc gctggggccg ccgcgtgcga
     3181 atctggtggc accttcgcgc ctgtctcgct gctttcgata agtctctagc catttaaaat
     3241 ttttgatgac ctgctgcgac gctttttttc tggcaagata gtcttgtaaa tgcgggccaa
     3301 gatctgcaca ctggtatttc ggtttttggg gccgcgggcg gcgacggggc ccgtgcgtcc
     3361 cagcgcacat gttcggcgag gcggggcctg cgagcgcggc caccgagaat cggacggggg
     3421 tagtctcaag ctggccggcc tgctctggtg cctggcctcg cgccgccgtg tatcgccccg
     3481 ccctgggcgg caaggctggc ccggtcggca ccagttgcgt gagcggaaag atggccgctt
     3541 cccggccctg ctgcagggag ctcaaaatgg aggacgcggc gctcgggaga gcgggcgggt
     3601 gagtcaccca cacaaaggaa aagggccttt ccgtcctcag ccgtcgcttc atgtgactcc
     3661 acggagtacc gggcgccgtc caggcacctc gattagttct cgagcttttg gagtacgtcg
     3721 tctttaggtt ggggggaggg gttttatgcg atggagtttc cccacactga gtgggtggag
     3781 actgaagtta ggccagcttg gcacttgatg taattctcct tggaatttgc cctttttgag
     3841 tttggatctt ggttcattct caagcctcag acagtggttc aaagtttttt tcttccattt
     3901 caggtgtcgt gagagctcgt ttagtgaacc gtcagatcgc ctggagacgc catccacgct
     3961 gttttgacct ccatagaaga cacacaagtt tgtacaaaaa agctgaacga gaaacgtaaa
     4021 atgatataaa tatcaatata ttaaattaga ttttgcataa aaaacagact acataatact
     4081 gtaaaacaca acatatccag tcactatggc gacagaagaa gtatagggat aacagggtaa
     4141 ttgttgtaag cgcgctatga tggaggctat gccactagaa tctgcgttcg ctaccttagg
     4201 accgttatag ttagaaggaa agctccatca tagtgactgg atatgttgtg ttttacagta
     4261 ttatgtagtc tgttttttat gcaaaatcta atttaatata ttgatattta tatcatttta
     4321 cgtttctcgt tcagctttct tgtacaaagt ggtaggagtg caggtggaaa ccatctcccc
     4381 aggagatggg cgcaccttcc ccaagcgcgg ccagacctgc gtggtgcact acaccgggat
     4441 gcttggagat ggaaagaaag ttgactcctc ccgggacaga aacaagccct ttaagtttat
     4501 gctaggcaag caggaggtga tccgaggctg ggaagaaggg gttgcccaga tgagtgtggg
     4561 tcagggagcc aaactgacta tatctccaga ttatgcctat ggtgccactg ggcacccagg
     4621 catcatccca ccacatgcca ctctcgtgtt cgatgtggag cttctagaac tggaatgaga
     4681 aatactcaga agatgtcact caccatagag agaccattaa tgggtctcct aggccactga
     4741 gcaagaacta tgaatgagat ctttagacat attgcggccg ctttctccgc gatccagaca
     4801 tgataagata cattgatgag tttggacaaa ccaaaactag aatgcagtga aaaaaatgcc
     4861 ttatttgtga aatttgtgat gctattgcct tatttgtaac cattataagc tgcaataaac
     4921 aagttatcta taacaagaaa atatatatat aataagttat cacgtaagta gaacatgaaa
     4981 taacaatata attatcgtat gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt
     5041 cattttgact cacgcggtcg ttatagttca aaatcagtga cacttaccgc attgacaagc
     5101 acgcctcacg ggagctccaa gcggcgactg agatgtccta aatgcacagc gacggattcg
     5161 cgctatttag aaagagagag caatatttca agaatgcatg cgtcaatttt acgcagacta
     5221 tctttctagg gttaaaaaaa gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa
     5281 ttgttatccg ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg
     5341 gggtgcctaa tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca
     5401 gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg
     5461 tttgcgtatt gggcgcactt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg
     5521 gctgcggcga gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg
     5581 ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa
     5641 ggccgcgttg ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg
     5701 acgctcaagt cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc
     5761 tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc
     5821 ctttctccct tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc
     5881 ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
     5941 ctgcgcctta tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc
     6001 actggcagca gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga
     6061 gttcttgaag tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc
     6121 tctgctgaag ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac
     6181 caccgctggt agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg
     6241 atctcaagaa gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc
     6301 acgttaaggg attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa
     6361 ttaaaaatga agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta
     6421 ccaatgctta atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt
     6481 tgcctgactc cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag
     6541 tgctgcaatg ataccgcgtg acccacgctc accggctcca gatttatcag caataaacca
     6601 gccagccgga agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
     6661 tattaattgt tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt
     6721 tgttgccatt gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag
     6781 ctccggttcc caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt
     6841 tagctccttc ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat
     6901 ggttatggca gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt
     6961 gactggtgag tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc
     7021 ttgcccggcg tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat
     7081 cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag
     7141 ttcgatgtaa cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt
     7201 ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg
     7261 gaaatgttga atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta
     7321 ttgtctcatg agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc
     7381 gcgcacattt ccccgaaaag tgccacctga cgtctaagaa accattatta tcatgacatt
     7441 aacctataaa aataggcgta tcacgaggcc ctttcgtc
//
