LOCUS       sequence-345098-        6241 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one piggyBac transposon destination vector for hEF1a 
            expression of Mega Gate cloned elements and hU6 expression of Golden
            Gate cloned guide RNAs.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6241)
  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 6241)
  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: PB-hEF1A-MG-U6-sgRNA
FEATURES             Location/Qualifiers
     source          1..6241
                     /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)"
     promoter        1423..2605
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1654..2596
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2553..2573
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     2607..2631
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    2677..2801
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(2922..3046)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             3048..3074
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    3196..3330
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     3274..3293
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     promoter        3339..3587
                     /label=U6 promoter
                     /note="RNA polymerase III promoter for human U6 snRNA 
                     (Domitrovich & Kunkel, 2003)"
     primer_bind     3339..3359
                     /label=hU6-F
                     /note="Human U6 promoter, forward primer"
     primer_bind     3510..3529
                     /label=LKO.1 5'
                     /note="Human U6 promoter, forward primer"
     misc_RNA        3606..3681
                     /label=gRNA scaffold
                     /note="guide RNA scaffold for the Streptococcus pyogenes 
                     CRISPR/Cas9 system"
     repeat_region   3960..3994
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4020..4036)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4020..4036)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4033..4055)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4044..4060
                     /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(4068..4074,4075..4092,4093..4098))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4113..4134
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4251..4268)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4422..5010)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4502..4521)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5181..5972,5973..6041))
                     /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     5804..5823
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6042..6146)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6214..6232
                     /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 tagcttaacc tatacataat agaaacaaat tatacttatt agtcagtcag
      781 aaacaacttt ggcacatatc aatattatgc tctcgttaat taactcgagg gatccggcgc
      841 gcctgtcatt ctaaatctct ctttcagcct aaagcttttt ccccgtatcc ccccaggtgt
      901 ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc
      961 cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg cggggggagc gccggaccgg
     1021 agcggagccc cgggcggctc gctgctgccc cctagcgggg gagggacgta attacatccc
     1081 tgggggcttt gggagggggc tgtccccgtg agctcccagg cgcgcctgtc attctaaatc
     1141 tctctttcag cctaaagctt tttccccgta tccccccagg tgtctgcagg ctcaaagagc
     1201 agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc
     1261 cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg
     1321 ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg
     1381 ggctgtcccc gtgagctccc aggcgcgccg ctagcttcgt gaggctccgg tgcccgtcag
     1441 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
     1501 accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
     1561 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
     1621 ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg
     1681 cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac gcccctggct
     1741 gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc
     1801 ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcttg ggcgctgggg
     1861 ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct
     1921 agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt
     1981 aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg
     2041 ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag
     2101 aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc
     2161 gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga
     2221 aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg
     2281 agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc
     2341 ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt
     2401 ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac
     2461 tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt
     2521 tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt
     2581 ttttcttcca tttcaggtgt cgtgagagct cgtttagtga accgtcagat cgcctggaga
     2641 cgccatccac gctgttttga cctccataga agacacacaa gtttgtacaa aaaagctgaa
     2701 cgagaaacgt aaaatgatat aaatatcaat atattaaatt agattttgca taaaaaacag
     2761 actacataat actgtaaaac acaacatatc cagtcactat ggcgacagaa gaagtatagg
     2821 gataacaggg taattgttgt aagcgcgcta tgatggaggc tatgccacta gaatctgcgt
     2881 tcgctacctt aggaccgtta tagttagaag gaaagctcca tcatagtgac tggatatgtt
     2941 gtgttttaca gtattatgta gtctgttttt tatgcaaaat ctaatttaat atattgatat
     3001 ttatatcatt ttacgtttct cgttcagctt tcttgtacaa agtggtatac ccatacgatg
     3061 ttccagatta cgcttagtaa tgagaaatac tcagaagatg tcactcacca tagagagacc
     3121 attaatgggt ctcctaggcc actgagcaag aactatgaat gagatcttta gacatattgc
     3181 ggccgctttc tccgcgatcc agacatgata agatacattg atgagtttgg acaaaccaaa
     3241 actagaatgc agtgaaaaaa atgccttatt tgtgaaattt gtgatgctat tgccttattt
     3301 gtaaccatta taagctgcaa taaacaagtt ggcaggaaga gggcctattt cccatgattc
     3361 cttcatattt gcatatacga tacaaggctg ttagagagat aattggaatt aatttgactg
     3421 taaacacaaa gatattagta caaaatacgt gacgtagaaa gtaataattt cttgggtagt
     3481 ttgcagtttt aaaattatgt tttaaaatgg actatcatat gcttaccgta acttgaaagt
     3541 atttcgattt cttggcttta tatatcttgt ggaaaggacg aaacaccgga agagcgagct
     3601 cttctgtttt agagctagaa atagcaagtt aaaataaggc tagtccgtta tcaacttgaa
     3661 aaagtggcac cgagtcggtg ctttttttat ctataacaag aaaatatata tataataagt
     3721 tatcacgtaa gtagaacatg aaataacaat ataattatcg tatgagttaa atcttaaaag
     3781 tcacgtaaaa gataatcatg cgtcattttg actcacgcgg tcgttatagt tcaaaatcag
     3841 tgacacttac cgcattgaca agcacgcctc acgggagctc caagcggcga ctgagatgtc
     3901 ctaaatgcac agcgacggat tcgcgctatt tagaaagaga gagcaatatt tcaagaatgc
     3961 atgcgtcaat tttacgcaga ctatctttct agggttaaaa aaagcttggc gtaatcatgg
     4021 tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa catacgagcc
     4081 ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac attaattgcg
     4141 ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca ttaatgaatc
     4201 ggccaacgcg cggggagagg cggtttgcgt attgggcgca cttccgcttc ctcgctcact
     4261 gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc aaaggcggta
     4321 atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc aaaaggccag
     4381 caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag gctccgcccc
     4441 cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc gacaggacta
     4501 taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg
     4561 ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcatagc
     4621 tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg ctgtgtgcac
     4681 gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct tgagtccaac
     4741 ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat tagcagagcg
     4801 aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg ctacactaga
     4861 agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt
     4921 agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
     4981 cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc tacggggtct
     5041 gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt atcaaaaagg
     5101 atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta aagtatatat
     5161 gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc
     5221 tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg
     5281 gagggcttac catctggccc cagtgctgca atgataccgc gtgacccacg ctcaccggct
     5341 ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag tggtcctgca
     5401 actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt aagtagttcg
     5461 ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt gtcacgctcg
     5521 tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt tacatgatcc
     5581 cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag
     5641 ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
     5701 ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt ctgagaatag
     5761 tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac cgcgccacat
     5821 agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa actctcaagg
     5881 atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa ctgatcttca
     5941 gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca
     6001 aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
     6061 tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga atgtatttag
     6121 aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccacc tgacgtctaa
     6181 gaaaccatta ttatcatgac attaacctat aaaaataggc gtatcacgag gccctttcgt
     6241 c
//
