LOCUS       sequence-345096-        6753 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one piggyBac transposon destination vector for 
            mCMV+dox-inducible expression of Mega Gate cloned elements 
            (CMVe-hEF1a-driven rtTA and puromycin resistance).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6753)
  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 6753)
  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-CT3G-CERP2-MG
FEATURES             Location/Qualifiers
     source          1..6753
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     protein_bind    17..35
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    53..71
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    89..107
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    125..143
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    161..179
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    197..215
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    233..251
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     278..302
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     protein_bind    368..492
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(613..737)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             739..765
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     polyA_signal    887..1021
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1293..1327
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1338..1355)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1338..1354)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1347..1369)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1563..1582)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1684..1706
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1745..1763)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1831..1935
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1936..2004,2005..2796)
                     /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     complement(2154..2173)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2967..3555
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3456..3475
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3709..3726
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3932..3953
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3968..3973,3974..3991,3992..3998)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4006..4022
                     /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)."
     primer_bind     4011..4033
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4030..4046
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4030..4046
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4072..4134
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4310..4391
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5108..5164)
                     /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             complement(5174..5917)
                     /codon_start=1
                     /product="modified rtTA protein that binds tightly to 
                     promoters containing the tet operator in the presence of 
                     doxycycline"
                     /label=Tet-On(R) 3G
                     /translation="MSRLDKSKVINSALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHSCPLEGESWQDFLRNNAKSYRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLKQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPTDALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     CDS             complement(5918..5941)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      5941..5950
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5970..6181)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        6182..6561
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
ORIGIN
        1 atcacctcga gtttactccc tatcagtgat agagaacgta tgaagagttt actccctatc
       61 agtgatagag aacgtatgca gactttactc cctatcagtg atagagaacg tataaggagt
      121 ttactcccta tcagtgatag agaacgtatg accagtttac tccctatcag tgatagagaa
      181 cgtatctaca gtttactccc tatcagtgat agagaacgta tatccagttt actccctatc
      241 agtgatagag aacgtataag cttgcctata taagcagagc tcgtttagtg aaccgtcaga
      301 tcgcctggag acgccatcca cgctgttttg acctccatag aagacaccgg gaccgatcca
      361 gcctaccaca agtttgtaca aaaaagctga acgagaaacg taaaatgata taaatatcaa
      421 tatattaaat tagattttgc ataaaaaaca gactacataa tactgtaaaa cacaacatat
      481 ccagtcacta tggcgacaga agaagtatag ggataacagg gtaattgttg taagcgcgct
      541 atgatggagg ctatgccact agaatctgcg ttcgctacct taggaccgtt atagttagaa
      601 ggaaagctcc atcatagtga ctggatatgt tgtgttttac agtattatgt agtctgtttt
      661 ttatgcaaaa tctaatttaa tatattgata tttatatcat tttacgtttc tcgttcagct
      721 ttcttgtaca aagtggtata cccatacgat gttccagatt acgcttagta atgagaaata
      781 ctcagaagat gtcactcacc atagagagac cattaatggg tctcctaggc cactgagcaa
      841 gaactatgaa tgagatcttt agacatattg cggccgcttt ctccgcgatc cagacatgat
      901 aagatacatt gatgagtttg gacaaaccaa aactagaatg cagtgaaaaa aatgccttat
      961 ttgtgaaatt tgtgatgcta ttgccttatt tgtaaccatt ataagctgca ataaacaagt
     1021 tatctataac aagaaaatat atatataata agttatcacg taagtagaac atgaaataac
     1081 aatataatta tcgtatgagt taaatcttaa aagtcacgta aaagataatc atgcgtcatt
     1141 ttgactcacg cggtcgttat agttcaaaat cagtgacact taccgcattg acaagcacgc
     1201 ctcacgggag ctccaagcgg cgactgagat gtcctaaatg cacagcgacg gattcgcgct
     1261 atttagaaag agagagcaat atttcaagaa tgcatgcgtc aattttacgc agactatctt
     1321 tctagggtta agaattcact ggccgtcgtt ttacaacgtc gtgactggga aaaccctggc
     1381 gttacccaac ttaatcgcct tgcagcacat ccccctttcg ccagctggcg taatagcgaa
     1441 gaggcccgca ccgatcgccc ttcccaacag ttgcgcagcc tgaatggcga atggcgcctg
     1501 atgcggtatt ttctccttac gcatctgtgc ggtatttcac accgcatatg gtgcactctc
     1561 agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct
     1621 gacgcgccct gacgggcttg tctgctcccg gcatccgctt acagacaagc tgtgaccatg
     1681 tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacac gcgagttcga
     1741 aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag
     1801 acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa
     1861 atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat
     1921 tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg
     1981 gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa
     2041 gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt
     2101 gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt
     2161 ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg catacactat
     2221 tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg
     2281 acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta
     2341 cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat
     2401 catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag
     2461 cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt aactggcgaa
     2521 ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca
     2581 ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc
     2641 ggtgagcgtg gttcacgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt
     2701 atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc
     2761 gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat
     2821 atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt
     2881 tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac
     2941 cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc
     3001 ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca
     3061 actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgttcttcta
     3121 gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct
     3181 ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg
     3241 gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc
     3301 acacagccca gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta
     3361 tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg
     3421 gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt
     3481 cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg
     3541 cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg
     3601 ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc
     3661 gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg
     3721 agcgaggaag cggatgagcg cccaatacgc aaaccgcctc tcaccgcgcg ttggccgatt
     3781 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca
     3841 aaccgcctct ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg
     3901 actggaaagc gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac
     3961 cccaggcttt acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac
     4021 aatttcacac aggaaacagc tatgaccatg attacgccaa ggtcgactta accctagaaa
     4081 gataatcata ttgtgacgta cgttaaagat aatcatgcgt aaaattgacg catgtgtttt
     4141 atcggtctgt atatcgaggt ttatttatta atttgaatag atattaagtt ttattatatt
     4201 tacacttaca tactaataat aaattcaaca aacaatttat ttatgtttat ttatttatta
     4261 aaaaaaaaca aaaactcaaa atttcttcta taaagtaaca aaacttttag cagtgaaaaa
     4321 aatgctttat ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca
     4381 ataaacaagt taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt
     4441 gggaggtttt ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatcctc
     4501 tggagattca tgcgccaggt tttctggtca tgcaccacgt tcgtgggccc tcaggaacct
     4561 ccacgtcagc ggtaactgtg aagcccagtc gctcgtaaaa aggaagattc cggggagcac
     4621 tggtttcgag gaacgccggc acgccagccc gttccgcggc ctcaacacca ggaagaacga
     4681 cagcgcttcc caaaccttta ccttggtgat ctgggctgac accgacagtg gcaagaaacc
     4741 acgcaggctc ctttggtcta tgaggagcca gcaagccctc catttgctgt tgtgccgcca
     4801 acctactccc tgacagctcg gccatccgcg gacctatttc cgcgaacact gcgccggctt
     4861 cgacgctttc cggagtagtc cagaccgcaa ctgcggcacc atcgtccgca acccaaactt
     4921 taccaatgtc cagacccacg cgcgtaagaa acagctcttg caactcagtt accctctcta
     4981 tgtgtctgtc cggatctacg gtatggcggg tcgctggata atctgcaaat gctgcggcaa
     5041 gtgttcggac agcccgtggt acatcgtcac gggtagcgag cctaacagtg ggcttatatt
     5101 ccgtcatggg gccggggttc tcctccacgt cgccggcctg cttcagcagg ctgaagttgg
     5161 tggcgccgct gcccccgggg agcatgtcaa ggtcaaaatc gtcaagagcg tcagcaggca
     5221 gcatatcaag gtcaaagtcg tcaagggcat cggctgggag catgtctaag tcaaaatcgt
     5281 caagggcgtc ggtcggcccg ccgctttcgc actttagctg tttctccagg ccacatatga
     5341 ttagttccag gccgaaaagg aaggcaggtt cggctccctg ccggtcgaac agctcaattg
     5401 cttgtttcag aagtgggggc atagaatcgg tggtaggtgt ctctctttcc tcttttgcta
     5461 cttgatgctc ctgttcctcc aatacgcagc ccagtgtaaa gtggcccacg gcggacagag
     5521 cgtacagtgc gttctccagg gagaagcctt gctgacacag gaacgcgagc tgattttcca
     5581 gggtttcgta ctgtttctct gttgggcggg tgccgagatg cactttagcc ccgtcacgat
     5641 gtgagaggag agcacagcgg tatgacttgg cgttgttccg cagaaagtct tgccatgact
     5701 cgccttccag ggggcaggag tgggtatgat gcctgtccag catctcgatt ggcagggcat
     5761 cgagcagggc ccgcttgttc ttcacgtgcc agtacagggt aggctgctca actcccagct
     5821 tttgagcgag tttccttgtc gtcaggcctt cgataccgac tccattgagt aattccagag
     5881 cagagtttat gactttgctc ttgtccagtc tagacatctt atcgtcatcg tctttgtaat
     5941 ccatggtggc ggatcccgcg tcacgacacc tgtgttctgg cggcaaaccc gttgcgaaaa
     6001 agaacgttca cggcgactac tgcacttata tacggttctc ccccaccctc gggaaaaagg
     6061 cggagccagt acacgacatc actttcccag tttaccccgc gccaccttct ctaggcaccg
     6121 gttcaattgc cgacccctcc ccccaacttc tcgggcactg tgggccatgt gcgctctgcc
     6181 ccatggtaat agccatgact aatacgtaga tgtactgcca agtaggaaag tgccataagg
     6241 tcatgtactg ggcataatgc caggcgggcc atttaccgtc attgacgtca atagggggcg
     6301 tacttggcat atgatacact tgatgtactg ccaagtgggc agtttaccgt aaatactcca
     6361 cccattgacg tcaatggaaa gtgcctattg gcgttactat gggaacatac gtcattattg
     6421 acgtcaatgg gcgggggtcg ttgggcggtc agccaggcgg gccatttacc gtaagttatg
     6481 taacgcggaa ctccatatat gggctatgaa ctaatgaccc cgtaattgat tactattaat
     6541 aactagtcaa taatcaatgt cataagctct aatcgcataa tcgagctaag agcgattaat
     6601 atgcttagct agcttagcta agctagattt ctctattctc ccacaatata tagcagctat
     6661 tagcttagca gtccgaatac gatcgtccac gatccataag cgtaacacat ttgagtagcg
     6721 actgacgggc accggagcca ctcgagtgga att
//
