LOCUS       sequence-339943-        7190 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  MegaGate destination with C-terminal HA tag and hygromycin 
            resistance.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7190)
  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 7190)
  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-HA-Hygro
FEATURES             Location/Qualifiers
     source          1..7190
                     /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..4381
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    4503..4637
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     4581..4600
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     repeat_region   4909..4943
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4969..4985)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4969..4985)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4982..5004)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4993..5009
                     /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(5017..5023,5024..5041,5042..5047))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5062..5083
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5200..5217)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5371..5959)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5451..5470)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6130..6921,6922..6990))
                     /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     6753..6772
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6991..7095)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     7163..7181
                     /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 tgtagtgtat
     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 ggtataccca tacgatgttc cagattacgc
     4381 ttagtaatga gaaatactca gaagatgtca ctcaccatag agagaccatt aatgggtctc
     4441 ctaggccact gagcaagaac tatgaatgag atctttagac atattgcggc cgctttctcc
     4501 gcgatccaga catgataaga tacattgatg agtttggaca aaccaaaact agaatgcagt
     4561 gaaaaaaatg ccttatttgt gaaatttgtg atgctattgc cttatttgta accattataa
     4621 gctgcaataa acaagttatc tataacaaga aaatatatat ataataagtt atcacgtaag
     4681 tagaacatga aataacaata taattatcgt atgagttaaa tcttaaaagt cacgtaaaag
     4741 ataatcatgc gtcattttga ctcacgcggt cgttatagtt caaaatcagt gacacttacc
     4801 gcattgacaa gcacgcctca cgggagctcc aagcggcgac tgagatgtcc taaatgcaca
     4861 gcgacggatt cgcgctattt agaaagagag agcaatattt caagaatgca tgcgtcaatt
     4921 ttacgcagac tatctttcta gggttaaaaa aagcttggcg taatcatggt catagctgtt
     4981 tcctgtgtga aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa
     5041 gtgtaaagcc tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact
     5101 gcccgctttc cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc
     5161 ggggagaggc ggtttgcgta ttgggcgcac ttccgcttcc tcgctcactg actcgctgcg
     5221 ctcggtcgtt cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc
     5281 cacagaatca ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag
     5341 gaaccgtaaa aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca
     5401 tcacaaaaat cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca
     5461 ggcgtttccc cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg
     5521 atacctgtcc gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag
     5581 gtatctcagt tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt
     5641 tcagcccgac cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca
     5701 cgacttatcg ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg
     5761 cggtgctaca gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt
     5821 tggtatctgc gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc
     5881 cggcaaacaa accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg
     5941 cagaaaaaaa ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg
     6001 gaacgaaaac tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta
     6061 gatcctttta aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg
     6121 gtctgacagt taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg
     6181 ttcatccata gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc
     6241 atctggcccc agtgctgcaa tgataccgcg tgacccacgc tcaccggctc cagatttatc
     6301 agcaataaac cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc
     6361 ctccatccag tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag
     6421 tttgcgcaac gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat
     6481 ggcttcattc agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg
     6541 caaaaaagcg gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt
     6601 gttatcactc atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag
     6661 atgcttttct gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg
     6721 accgagttgc tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt
     6781 aaaagtgctc atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct
     6841 gttgagatcc agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac
     6901 tttcaccagc gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat
     6961 aagggcgaca cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat
     7021 ttatcagggt tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca
     7081 aataggggtt ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
     7141 tatcatgaca ttaacctata aaaataggcg tatcacgagg ccctttcgtc
//
