LOCUS       sequence-345872-        7519 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 upstream 
            of IRES2-mNeonGreen (hEF1a-driven rtTA and puromycin resistance).
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7519)
  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 7519)
  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-ERP2-MG-IRES2-mNeonGreen
FEATURES             Location/Qualifiers
     source          1..7519
                     /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    386..510
                     /gene="mutant version of attR"
                     /label=attR1
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     protein_bind    complement(631..755)
                     /gene="mutant version of attR"
                     /label=attR2
                     /bound_moiety="LR Clonase(TM)"
                     /note="recombination site for the Gateway(R) LR reaction"
     CDS             757..783
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     misc_feature    join(877..1451,1452..1454,1455..1463)
                     /label=IRES2
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(1044..1061)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     1271..1290
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     polyA_signal    2192..2326
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2598..2632
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2643..2660)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2643..2659)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2652..2674)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2868..2887)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2989..3011
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3050..3068)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3136..3240
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3241..3309,3310..4101)
                     /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(3459..3478)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4272..4860
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4761..4780
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5014..5031
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5237..5258
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5273..5278,5279..5296,5297..5303)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5311..5327
                     /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     5316..5338
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5335..5351
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5335..5351
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   5377..5439
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    5615..5696
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(6413..6469)
                     /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(6479..7222)
                     /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(7223..7246)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      7246..7255
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(7275..7486)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
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 gcctccgcgg tggcggcatc ggtacacaag tttgtacaaa aaagctgaac gagaaacgta
      421 aaatgatata aatatcaata tattaaatta gattttgcat aaaaaacaga ctacataata
      481 ctgtaaaaca caacatatcc agtcactatg gcgacagaag aagtataggg ataacagggt
      541 aattgttgta agcgcgctat gatggaggct atgccactag aatctgcgtt cgctacctta
      601 ggaccgttat agttagaagg aaagctccat catagtgact ggatatgttg tgttttacag
      661 tattatgtag tctgtttttt atgcaaaatc taatttaata tattgatatt tatatcattt
      721 tacgtttctc gttcagcttt cttgtacaaa gtggtatacc catacgatgt tccagattac
      781 gcttagtaat gagaaatact cagaagatgt cactcaccat agagagacca ttaatgggtc
      841 tcctaggcca ctgagcaaga actatgaatg agatctcccc tctccctccc ccccccctaa
      901 cgttactggc cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc
      961 caccatattg ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac
     1021 gagcattcct aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt
     1081 gaaggaagca gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg
     1141 caggcagcgg aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata
     1201 agatacacct gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga
     1261 aagagtcaaa tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt
     1321 accccattgt atgggatctg atctggggcc tcggtacaca tgctttacat gtgtttagtc
     1381 gaggttaaaa aaacgtctag gccccccgaa ccacggggac gtggttttcc tttgaaaaac
     1441 acgatgataa tatggccaca accatggtca gcaaaggcga agaagataac atggcatcac
     1501 tgccagcgac acacgaactt catattttcg gctccatcaa tggtgtggac ttcgatatgg
     1561 ttggacaggg tactgggaac cctaacgatg gatatgagga gttgaacttg aaaagtacca
     1621 agggcgatct tcagttttca ccttggattc tcgtacctca tataggttat ggctttcatc
     1681 aatatctgcc ctatcctgac ggtatgtcac ctttccaagc agctatggtt gacgggagcg
     1741 gttatcaggt tcaccggaca atgcagtttg aagatggagc ctctttgacc gtaaattatc
     1801 ggtatactta tgagggcagc catattaaag gcgaggctca agttaaggga actgggttcc
     1861 ccgcggatgg tcctgtaatg acaaacagtc tgacggctgc agactggtgc cgctctaaga
     1921 aaacatatcc aaatgacaag acgattatta gcactttcaa gtggtcatac actacaggaa
     1981 atggtaaacg ctataggagc actgcccgga ccacgtatac tttcgcgaag ccgatggcgg
     2041 caaattattt gaaaaaccaa ccgatgtatg tgttcagaaa gacagaactc aagcactcta
     2101 aaactgaact gaacttcaaa gagtggcaga aagcctttac tgatgtaatg ggcatggacg
     2161 aactctataa gtaagcggcc gctttctccg cgatccagac atgataagat acattgatga
     2221 gtttggacaa accaaaacta gaatgcagtg aaaaaaatgc cttatttgtg aaatttgtga
     2281 tgctattgcc ttatttgtaa ccattataag ctgcaataaa caagttatct ataacaagaa
     2341 aatatatata taataagtta tcacgtaagt agaacatgaa ataacaatat aattatcgta
     2401 tgagttaaat cttaaaagtc acgtaaaaga taatcatgcg tcattttgac tcacgcggtc
     2461 gttatagttc aaaatcagtg acacttaccg cattgacaag cacgcctcac gggagctcca
     2521 agcggcgact gagatgtcct aaatgcacag cgacggattc gcgctattta gaaagagaga
     2581 gcaatatttc aagaatgcat gcgtcaattt tacgcagact atctttctag ggttaagaat
     2641 tcactggccg tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat
     2701 cgccttgcag cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat
     2761 cgcccttccc aacagttgcg cagcctgaat ggcgaatggc gcctgatgcg gtattttctc
     2821 cttacgcatc tgtgcggtat ttcacaccgc atatggtgca ctctcagtac aatctgctct
     2881 gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg
     2941 gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccatgtctcc gggagctgca
     3001 tgtgtcagag gttttcaccg tcatcaccga aacacgcgag ttcgaaaggg cctcgtgata
     3061 cgcctatttt tataggttaa tgtcatgata ataatggttt cttagacgtc aggtggcact
     3121 tttcggggaa atgtgcgcgg aacccctatt tgtttatttt tctaaataca ttcaaatatg
     3181 tatccgctca tgagacaata accctgataa atgcttcaat aatattgaaa aaggaagagt
     3241 atgagtattc aacatttccg tgtcgccctt attccctttt ttgcggcatt ttgccttcct
     3301 gtttttgctc acccagaaac gctggtgaaa gtaaaagatg ctgaagatca gttgggtgca
     3361 cgagtgggtt acatcgaact ggatctcaac agcggtaaga tccttgagag ttttcgcccc
     3421 gaagaacgtt ttccaatgat gagcactttt aaagttctgc tatgtggcgc ggtattatcc
     3481 cgtattgacg ccgggcaaga gcaactcggt cgccgcatac actattctca gaatgacttg
     3541 gttgagtact caccagtcac agaaaagcat cttacggatg gcatgacagt aagagaatta
     3601 tgcagtgctg ccataaccat gagtgataac actgcggcca acttacttct gacaacgatc
     3661 ggaggaccga aggagctaac cgcttttttg cacaacatgg gggatcatgt aactcgcctt
     3721 gatcgttggg aaccggagct gaatgaagcc ataccaaacg acgagcgtga caccacgatg
     3781 cctgtagcaa tggcaacaac gttgcgcaaa ctattaactg gcgaactact tactctagct
     3841 tcccggcaac aattaataga ctggatggag gcggataaag ttgcaggacc acttctgcgc
     3901 tcggcccttc cggctggctg gtttattgct gataaatctg gagccggtga gcgtggttca
     3961 cgcggtatca ttgcagcact ggggccagat ggtaagccct cccgtatcgt agttatctac
     4021 acgacgggga gtcaggcaac tatggatgaa cgaaatagac agatcgctga gataggtgcc
     4081 tcactgatta agcattggta actgtcagac caagtttact catatatact ttagattgat
     4141 ttaaaacttc atttttaatt taaaaggatc taggtgaaga tcctttttga taatctcatg
     4201 accaaaatcc cttaacgtga gttttcgttc cactgagcgt cagaccccgt agaaaagatc
     4261 aaaggatctt cttgagatcc tttttttctg cgcgtaatct gctgcttgca aacaaaaaaa
     4321 ccaccgctac cagcggtggt ttgtttgccg gatcaagagc taccaactct ttttccgaag
     4381 gtaactggct tcagcagagc gcagatacca aatactgttc ttctagtgta gccgtagtta
     4441 ggccaccact tcaagaactc tgtagcaccg cctacatacc tcgctctgct aatcctgtta
     4501 ccagtggctg ctgccagtgg cgataagtcg tgtcttaccg ggttggactc aagacgatag
     4561 ttaccggata aggcgcagcg gtcgggctga acggggggtt cgtgcacaca gcccagcttg
     4621 gagcgaacga cctacaccga actgagatac ctacagcgtg agctatgaga aagcgccacg
     4681 cttcccgaag ggagaaaggc ggacaggtat ccggtaagcg gcagggtcgg aacaggagag
     4741 cgcacgaggg agcttccagg gggaaacgcc tggtatcttt atagtcctgt cgggtttcgc
     4801 cacctctgac ttgagcgtcg atttttgtga tgctcgtcag gggggcggag cctatggaaa
     4861 aacgccagca acgcggcctt tttacggttc ctggcctttt gctggccttt tgctcacatg
     4921 ttctttcctg cgttatcccc tgattctgtg gataaccgta ttaccgcctt tgagtgagct
     4981 gataccgctc gccgcagccg aacgaccgag cgcagcgagt cagtgagcga ggaagcggat
     5041 gagcgcccaa tacgcaaacc gcctctcacc gcgcgttggc cgattcatta atgcagctgg
     5101 cacgacaggt ttcccgactg gaaagcgggc agtgagcgca acgcaaaccg cctctccccg
     5161 cgcgttggcc gattcattaa tgcagctggc acgacaggtt tcccgactgg aaagcgggca
     5221 gtgagcgcaa cgcaattaat gtgagttagc tcactcatta ggcaccccag gctttacact
     5281 ttatgcttcc ggctcgtatg ttgtgtggaa ttgtgagcgg ataacaattt cacacaggaa
     5341 acagctatga ccatgattac gccaaggtcg acttaaccct agaaagataa tcatattgtg
     5401 acgtacgtta aagataatca tgcgtaaaat tgacgcatgt gttttatcgg tctgtatatc
     5461 gaggtttatt tattaatttg aatagatatt aagttttatt atatttacac ttacatacta
     5521 ataataaatt caacaaacaa tttatttatg tttatttatt tattaaaaaa aaacaaaaac
     5581 tcaaaatttc ttctataaag taacaaaact tttagcagtg aaaaaaatgc tttatttgtg
     5641 aaatttgtga tgctattgct ttatttgtaa ccattataag ctgcaataaa caagttaaca
     5701 acaacaattg cattcatttt atgtttcagg ttcaggggga ggtgtgggag gttttttaaa
     5761 gcaagtaaaa cctctacaaa tgtggtatgg ctgattatga tcctctggag attcatgcgc
     5821 caggttttct ggtcatgcac cacgttcgtg ggccctcagg aacctccacg tcagcggtaa
     5881 ctgtgaagcc cagtcgctcg taaaaaggaa gattccgggg agcactggtt tcgaggaacg
     5941 ccggcacgcc agcccgttcc gcggcctcaa caccaggaag aacgacagcg cttcccaaac
     6001 ctttaccttg gtgatctggg ctgacaccga cagtggcaag aaaccacgca ggctcctttg
     6061 gtctatgagg agccagcaag ccctccattt gctgttgtgc cgccaaccta ctccctgaca
     6121 gctcggccat ccgcggacct atttccgcga acactgcgcc ggcttcgacg ctttccggag
     6181 tagtccagac cgcaactgcg gcaccatcgt ccgcaaccca aactttacca atgtccagac
     6241 ccacgcgcgt aagaaacagc tcttgcaact cagttaccct ctctatgtgt ctgtccggat
     6301 ctacggtatg gcgggtcgct ggataatctg caaatgctgc ggcaagtgtt cggacagccc
     6361 gtggtacatc gtcacgggta gcgagcctaa cagtgggctt atattccgtc atggggccgg
     6421 ggttctcctc cacgtcgccg gcctgcttca gcaggctgaa gttggtggcg ccgctgcccc
     6481 cggggagcat gtcaaggtca aaatcgtcaa gagcgtcagc aggcagcata tcaaggtcaa
     6541 agtcgtcaag ggcatcggct gggagcatgt ctaagtcaaa atcgtcaagg gcgtcggtcg
     6601 gcccgccgct ttcgcacttt agctgtttct ccaggccaca tatgattagt tccaggccga
     6661 aaaggaaggc aggttcggct ccctgccggt cgaacagctc aattgcttgt ttcagaagtg
     6721 ggggcataga atcggtggta ggtgtctctc tttcctcttt tgctacttga tgctcctgtt
     6781 cctccaatac gcagcccagt gtaaagtggc ccacggcgga cagagcgtac agtgcgttct
     6841 ccagggagaa gccttgctga cacaggaacg cgagctgatt ttccagggtt tcgtactgtt
     6901 tctctgttgg gcgggtgccg agatgcactt tagccccgtc gcgatgtgag aggagagcac
     6961 agcggtatga cttggcgttg ttccgcagaa agtcttgcca tgactcgcct tccagggggc
     7021 aggagtgggt atgatgcctg tccagcatct cgattggcag ggcatcgagc agggcccgct
     7081 tgttcttcac gtgccagtac agggtaggct gctcaactcc cagcttttga gcgagtttcc
     7141 ttgtcgtcag gccttcgata ccgactccat tgagtaattc cagagcagag tttatgactt
     7201 tgctcttgtc cagtctagac atcttatcgt catcgtcttt gtaatccatg gtggcggatc
     7261 ccgcgtcacg acacctgtgt tctggcggca aacccgttgc gaaaaagaac gttcacggcg
     7321 actactgcac ttatatacgg ttctccccca ccctcgggaa aaaggcggag ccagtacacg
     7381 acatcacttt cccagtttac cccgcgccac cttctctagg caccggttca attgccgacc
     7441 cctcccccca acttctcggg gactgtgggc gatgtgcgct ctgcccactg acgggcaccg
     7501 gagccactcg agtggaatt
//
