LOCUS       sequence-345877-        6497 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  All-in-one piggyBac transposon vector for mCMV+dox-inducible 
            expression of Golden 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 6497)
  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 6497)
  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
FEATURES             Location/Qualifiers
     source          1..6497
                     /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"
     polyA_signal    627..761
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1037..1071
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1082..1099)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1082..1098)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1091..1113)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1307..1326)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     1428..1450
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1489..1507)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1575..1679
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1680..1748,1749..2540)
                     /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(1898..1917)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2711..3299
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3200..3219
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3453..3470
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    3676..3697
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(3712..3717,3718..3735,3736..3742)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    3750..3766
                     /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     3755..3777
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     3774..3790
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     3774..3790
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   3816..3878
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4054..4135
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4852..4908)
                     /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(4918..5661)
                     /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(5662..5685)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      5685..5694
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(5714..5925)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     enhancer        5926..6305
                     /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 gcctccgcgg tggcggcatc ggtaccgcca cccgagacca ttaatgggtc tcgtaatgat
      421 aggcgtcttc gaataatgga agacatacta gtagaccacc tcccctgcga gctaagctgg
      481 acagccaatg acgggtaaga gagtgacatt tttcactaac ctaagacagg agggccgtca
      541 gagctactgc ctaatccaaa gacgggtaaa agtgataaaa atgtatcact ccaacctaag
      601 acaggcgcag cttccgaggg atttgagatc cagacatgat aagatacatt gatgagtttg
      661 gacaaaccaa aactagaatg cagtgaaaaa aatgccttat ttgtgaaatt tgtgatgcta
      721 ttgccttatt tgtaaccatt ataagctgca ataaacaagt ttgatatcta taacaagaaa
      781 atatatatat aataagttat cacgtaagta gaacatgaaa taacaatata attatcgtat
      841 gagttaaatc ttaaaagtca cgtaaaagat aatcatgcgt cattttgact cacgcggtcg
      901 ttatagttca aaatcagtga cacttaccgc attgacaagc acgcctcacg ggagctccaa
      961 gcggcgactg agatgtccta aatgcacagc gacggattcg cgctatttag aaagagagag
     1021 caatatttca agaatgcatg cgtcaatttt acgcagacta tctttctagg gttaagaatt
     1081 cactggccgt cgttttacaa cgtcgtgact gggaaaaccc tggcgttacc caacttaatc
     1141 gccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc
     1201 gcccttccca acagttgcgc agcctgaatg gcgaatggcg cctgatgcgg tattttctcc
     1261 ttacgcatct gtgcggtatt tcacaccgca tatggtgcac tctcagtaca atctgctctg
     1321 atgccgcata gttaagccag ccccgacacc cgccaacacc cgctgacgcg ccctgacggg
     1381 cttgtctgct cccggcatcc gcttacagac aagctgtgac catgtctccg ggagctgcat
     1441 gtgtcagagg ttttcaccgt catcaccgaa acacgcgagt tcgaaagggc ctcgtgatac
     1501 gcctattttt ataggttaat gtcatgataa taatggtttc ttagacgtca ggtggcactt
     1561 ttcggggaaa tgtgcgcgga acccctattt gtttattttt ctaaatacat tcaaatatgt
     1621 atccgctcat gagacaataa ccctgataaa tgcttcaata atattgaaaa aggaagagta
     1681 tgagtattca acatttccgt gtcgccctta ttcccttttt tgcggcattt tgccttcctg
     1741 tttttgctca cccagaaacg ctggtgaaag taaaagatgc tgaagatcag ttgggtgcac
     1801 gagtgggtta catcgaactg gatctcaaca gcggtaagat ccttgagagt tttcgccccg
     1861 aagaacgttt tccaatgatg agcactttta aagttctgct atgtggcgcg gtattatccc
     1921 gtattgacgc cgggcaagag caactcggtc gccgcataca ctattctcag aatgacttgg
     1981 ttgagtactc accagtcaca gaaaagcatc ttacggatgg catgacagta agagaattat
     2041 gcagtgctgc cataaccatg agtgataaca ctgcggccaa cttacttctg acaacgatcg
     2101 gaggaccgaa ggagctaacc gcttttttgc acaacatggg ggatcatgta actcgccttg
     2161 atcgttggga accggagctg aatgaagcca taccaaacga cgagcgtgac accacgatgc
     2221 ctgtagcaat ggcaacaacg ttgcgcaaac tattaactgg cgaactactt actctagctt
     2281 cccggcaaca attaatagac tggatggagg cggataaagt tgcaggacca cttctgcgct
     2341 cggcccttcc ggctggctgg tttattgctg ataaatctgg agccggtgag cgtggttcac
     2401 gcggtatcat tgcagcactg gggccagatg gtaagccctc ccgtatcgta gttatctaca
     2461 cgacggggag tcaggcaact atggatgaac gaaatagaca gatcgctgag ataggtgcct
     2521 cactgattaa gcattggtaa ctgtcagacc aagtttactc atatatactt tagattgatt
     2581 taaaacttca tttttaattt aaaaggatct aggtgaagat cctttttgat aatctcatga
     2641 ccaaaatccc ttaacgtgag ttttcgttcc actgagcgtc agaccccgta gaaaagatca
     2701 aaggatcttc ttgagatcct ttttttctgc gcgtaatctg ctgcttgcaa acaaaaaaac
     2761 caccgctacc agcggtggtt tgtttgccgg atcaagagct accaactctt tttccgaagg
     2821 taactggctt cagcagagcg cagataccaa atactgttct tctagtgtag ccgtagttag
     2881 gccaccactt caagaactct gtagcaccgc ctacatacct cgctctgcta atcctgttac
     2941 cagtggctgc tgccagtggc gataagtcgt gtcttaccgg gttggactca agacgatagt
     3001 taccggataa ggcgcagcgg tcgggctgaa cggggggttc gtgcacacag cccagcttgg
     3061 agcgaacgac ctacaccgaa ctgagatacc tacagcgtga gctatgagaa agcgccacgc
     3121 ttcccgaagg gagaaaggcg gacaggtatc cggtaagcgg cagggtcgga acaggagagc
     3181 gcacgaggga gcttccaggg ggaaacgcct ggtatcttta tagtcctgtc gggtttcgcc
     3241 acctctgact tgagcgtcga tttttgtgat gctcgtcagg ggggcggagc ctatggaaaa
     3301 acgccagcaa cgcggccttt ttacggttcc tggccttttg ctggcctttt gctcacatgt
     3361 tctttcctgc gttatcccct gattctgtgg ataaccgtat taccgccttt gagtgagctg
     3421 ataccgctcg ccgcagccga acgaccgagc gcagcgagtc agtgagcgag gaagcggatg
     3481 agcgcccaat acgcaaaccg cctctcaccg cgcgttggcc gattcattaa tgcagctggc
     3541 acgacaggtt tcccgactgg aaagcgggca gtgagcgcaa cgcaaaccgc ctctccccgc
     3601 gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga aagcgggcag
     3661 tgagcgcaac gcaattaatg tgagttagct cactcattag gcaccccagg ctttacactt
     3721 tatgcttccg gctcgtatgt tgtgtggaat tgtgagcgga taacaatttc acacaggaaa
     3781 cagctatgac catgattacg ccaaggtcga cttaacccta gaaagataat catattgtga
     3841 cgtacgttaa agataatcat gcgtaaaatt gacgcatgtg ttttatcggt ctgtatatcg
     3901 aggtttattt attaatttga atagatatta agttttatta tatttacact tacatactaa
     3961 taataaattc aacaaacaat ttatttatgt ttatttattt attaaaaaaa aacaaaaact
     4021 caaaatttct tctataaagt aacaaaactt ttagcagtga aaaaaatgct ttatttgtga
     4081 aatttgtgat gctattgctt tatttgtaac cattataagc tgcaataaac aagttaacaa
     4141 caacaattgc attcatttta tgtttcaggt tcagggggag gtgtgggagg ttttttaaag
     4201 caagtaaaac ctctacaaat gtggtatggc tgattatgat cctctggaga ttcatgcgcc
     4261 aggttttctg gtcatgcacc acgttcgtgg gccctcagga acctccacgt cagcggtaac
     4321 tgtgaagccc agtcgctcgt aaaaaggaag attccgggga gcactggttt cgaggaacgc
     4381 cggcacgcca gcccgttccg cggcctcaac accaggaaga acgacagcgc ttcccaaacc
     4441 tttaccttgg tgatctgggc tgacaccgac agtggcaaga aaccacgcag gctcctttgg
     4501 tctatgagga gccagcaagc cctccatttg ctgttgtgcc gccaacctac tccctgacag
     4561 ctcggccatc cgcggaccta tttccgcgaa cactgcgccg gcttcgacgc tttccggagt
     4621 agtccagacc gcaactgcgg caccatcgtc cgcaacccaa actttaccaa tgtccagacc
     4681 cacgcgcgta agaaacagct cttgcaactc agttaccctc tctatgtgtc tgtccggatc
     4741 tacggtatgg cgggtcgctg gataatctgc aaatgctgcg gcaagtgttc ggacagcccg
     4801 tggtacatcg tcacgggtag cgagcctaac agtgggctta tattccgtca tggggccggg
     4861 gttctcctcc acgtcgccgg cctgcttcag caggctgaag ttggtggcgc cgctgccccc
     4921 ggggagcatg tcaaggtcaa aatcgtcaag agcgtcagca ggcagcatat caaggtcaaa
     4981 gtcgtcaagg gcatcggctg ggagcatgtc taagtcaaaa tcgtcaaggg cgtcggtcgg
     5041 cccgccgctt tcgcacttta gctgtttctc caggccacat atgattagtt ccaggccgaa
     5101 aaggaaggca ggttcggctc cctgccggtc gaacagctca attgcttgtt tcagaagtgg
     5161 gggcatagaa tcggtggtag gtgtctctct ttcctctttt gctacttgat gctcctgttc
     5221 ctccaatacg cagcccagtg taaagtggcc cacggcggac agagcgtaca gtgcgttctc
     5281 cagggagaag ccttgctgac acaggaacgc gagctgattt tccagggttt cgtactgttt
     5341 ctctgttggg cgggtgccga gatgcacttt agccccgtca cgatgtgaga ggagagcaca
     5401 gcggtatgac ttggcgttgt tccgcagaaa gtcttgccat gactcgcctt ccagggggca
     5461 ggagtgggta tgatgcctgt ccagcatctc gattggcagg gcatcgagca gggcccgctt
     5521 gttcttcacg tgccagtaca gggtaggctg ctcaactccc agcttttgag cgagtttcct
     5581 tgtcgtcagg ccttcgatac cgactccatt gagtaattcc agagcagagt ttatgacttt
     5641 gctcttgtcc agtctagaca tcttatcgtc atcgtctttg taatccatgg tggcggatcc
     5701 cgcgtcacga cacctgtgtt ctggcggcaa acccgttgcg aaaaagaacg ttcacggcga
     5761 ctactgcact tatatacggt tctcccccac cctcgggaaa aaggcggagc cagtacacga
     5821 catcactttc ccagtttacc ccgcgccacc ttctctaggc accggttcaa ttgccgaccc
     5881 ctccccccaa cttctcgggc actgtgggcc atgtgcgctc tgccccatgg taatagccat
     5941 gactaatacg tagatgtact gccaagtagg aaagtgccat aaggtcatgt actgggcata
     6001 atgccaggcg ggccatttac cgtcattgac gtcaataggg ggcgtacttg gcatatgata
     6061 cacttgatgt actgccaagt gggcagttta ccgtaaatac tccacccatt gacgtcaatg
     6121 gaaagtgcct attggcgtta ctatgggaac atacgtcatt attgacgtca atgggcgggg
     6181 gtcgttgggc ggtcagccag gcgggccatt taccgtaagt tatgtaacgc ggaactccat
     6241 atatgggcta tgaactaatg accccgtaat tgattactat taataactag tcaataatca
     6301 atgtcataag ctctaatcgc ataatcgagc taagagcgat taatatgctt agctagctta
     6361 gctaagctag atttctctat tctcccacaa tatatagcag ctattagctt agcagtccga
     6421 atacgatcgt ccacgatcca taagcgtaac acatttgagt agcgactgac gggcaccgga
     6481 gccactcgag tggaatt
//
