LOCUS       sequence-467762-        6663 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Tet-On 3G-inducible PiggyBac system for expression of Cre in human 
            pluripotent stem cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6663)
  TITLE     Heat shock promoters in human pluripotent stem cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6663)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: XLone-Cre
FEATURES             Location/Qualifiers
     source          1..6663
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        9..376
                     /label=TRE3GS promoter
                     /note="3rd-generation Tet-responsive promoter that can be 
                     activated by binding of Tet-On(R) 3G, modified to eliminate
                     binding sites for endogenous mammalian transcription 
                     factors"
     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"
     regulatory      383..392
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             395..415
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             416..1444
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="SNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLL
                     SVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLP
                     RPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFL
                     GIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVER
                     WISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQR
                     YLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLED
                     GD"
     polyA_signal    1625..1759
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   2035..2069
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(2080..2097)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2080..2096)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2089..2111)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(2305..2324)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2424..2446
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2484..2502)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2570..2674
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2675..2743,2744..3535)
                     /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(2893..2912)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3706..4294
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4195..4214
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4448..4465
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4582..4603
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4618..4623,4624..4641,4642..4648)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4656..4672
                     /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     4661..4683
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4680..4696
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4680..4696
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4722..4784
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4960..5041
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(5158..5556)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(5557..5613)
                     /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(5623..6366)
                     /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(6367..6390)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6390..6399
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6419..6630)
                     /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 ctttgcttat gtaaaccagg gcgcctataa aagagtgctg
      301 attttttgag taaacttcaa ttccacaaca cttttgtctt ataccaactt tccgtaccac
      361 ttcctaccct cgtaaaggta ccgccaccat gggcccaaag aagaagagaa aggtttcgaa
      421 tttactgacc gtacaccaaa atttgcctgc attaccggtc gatgcaacga gtgatgaggt
      481 tcgcaagaac ctgatggaca tgttcaggga tcgccaggcg ttttctgagc atacctggaa
      541 aatgcttctg tccgtttgcc ggtcgtgggc ggcatggtgc aagttgaata accggaaatg
      601 gtttcccgca gaacctgaag atgttcgcga ttatcttcta tatcttcagg cgcgcggtct
      661 ggcagtaaaa actatccagc aacatttggg ccagctaaac atgcttcatc gtcggtccgg
      721 gctgccacga ccaagtgaca gcaatgctgt ttcactggtt atgcggcgga tccgaaaaga
      781 aaacgttgat gccggtgaac gtgcaaaaca ggctctagcg ttcgaacgca ctgatttcga
      841 ccaggttcgt tcactcatgg aaaatagcga tcgctgccag gatatacgta atctggcatt
      901 tctggggatt gcttataaca ccctgttacg tatagccgaa attgccagga tcagggttaa
      961 agatatctca cgtactgacg gtgggagaat gttaatccat attggcagaa cgaaaacgct
     1021 ggttagcacc gcaggtgtag agaaggcact tagcctgggg gtaactaaac tggtcgagcg
     1081 atggatttcc gtctctggtg tagctgatga tccgaataac tacctgtttt gccgggtcag
     1141 aaaaaatggt gttgccgcgc catctgccac cagccagcta tcaactcgcg ccctggaagg
     1201 gatttttgaa gcaactcatc gattgattta cggcgctaag gatgactctg gtcagagata
     1261 cctggcctgg tctggacaca gtgcccgtgt cggagccgcg cgagatatgg cccgcgctgg
     1321 agtttcaata ccggagatca tgcaagctgg tggctggacc aatgtaaata ttgtcatgaa
     1381 ctatatccgt aacctggata gtgaaacagg ggcaatggtg cgcctgctgg aagatggcga
     1441 ttagactagt agaccacctc ccctgcgagc taagctggac agccaatgac gggtaagaga
     1501 gtgacatttt tcactaacct aagacaggag ggccgtcaga gctactgcct aatccaaaga
     1561 cgggtaaaag tgataaaaat gtatcactcc aacctaagac aggcgcagct tccgagggat
     1621 ttgagatcca gacatgataa gatacattga tgagtttgga caaaccaaaa ctagaatgca
     1681 gtgaaaaaaa tgccttattt gtgaaatttg tgatgctatt gccttatttg taaccattat
     1741 aagctgcaat aaacaagttt gatatctata acaagaaaat atatatataa taagttatca
     1801 cgtaagtaga acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg
     1861 taaaagataa tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca
     1921 cttaccgcat tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa
     1981 tgcacagcga cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg
     2041 tcaattttac gcagactatc tttctagggt taagaattca ctggccgtcg ttttacaacg
     2101 tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac atcccccttt
     2161 cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac agttgcgcag
     2221 cctgaatggc gaatggcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc
     2281 acaccgcata tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc
     2341 ccgacacccg ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc
     2401 ttacagacaa gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc
     2461 accgaaacgc gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat
     2521 gataataatg gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc
     2581 tatttgttta tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg
     2641 ataaatgctt caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc
     2701 ccttattccc ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt
     2761 gaaagtaaaa gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct
     2821 caacagcggt aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac
     2881 ttttaaagtt ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact
     2941 cggtcgccgc atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa
     3001 gcatcttacg gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga
     3061 taacactgcg gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt
     3121 tttgcacaac atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga
     3181 agccatacca aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg
     3241 caaactatta actggcgaac tacttactct agcttcccgg caacaattaa tagactggat
     3301 ggaggcggat aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat
     3361 tgctgataaa tctggagccg gtgagcgtgg gtctcgcggt atcattgcag cactggggcc
     3421 agatggtaag ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga
     3481 tgaacgaaat agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc
     3541 agaccaagtt tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag
     3601 gatctaggtg aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc
     3661 gttccactga gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt
     3721 tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt
     3781 gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat
     3841 accaaatact gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc
     3901 accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa
     3961 gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg
     4021 ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag
     4081 atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag
     4141 gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa
     4201 cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt
     4261 gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg
     4321 gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat cccctgattc
     4381 tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca gccgaacgac
     4441 cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ccaatacgca aaccgcctct
     4501 ccccgcgcgt tggccgattc attaatgcag ctggcacgac aggtttcccg actggaaagc
     4561 gggcagtgag cgcaacgcaa ttaatgtgag ttagctcact cattaggcac cccaggcttt
     4621 acactttatg cttccggctc gtatgttgtg tggaattgtg agcggataac aatttcacac
     4681 aggaaacagc tatgaccatg attacgccaa ggtcgactta accctagaaa gataatcata
     4741 ttgtgacgta cgttaaagat aatcatgcgt aaaattgacg catgtgtttt atcggtctgt
     4801 atatcgaggt ttatttatta atttgaatag atattaagtt ttattatatt tacacttaca
     4861 tactaataat aaattcaaca aacaatttat ttatgtttat ttatttatta aaaaaaaaca
     4921 aaaactcaaa atttcttcta taaagtaaca aaacttttag cagtgaaaaa aatgctttat
     4981 ttgtgaaatt tgtgatgcta ttgctttatt tgtaaccatt ataagctgca ataaacaagt
     5041 taacaacaac aattgcattc attttatgtt tcaggttcag ggggaggtgt gggaggtttt
     5101 ttaaagcaag taaaacctct acaaatgtgg tatggctgat tatgatcctc tggagattta
     5161 gccctcccac acataaccag agggcagcaa ttcacgaatc ccaactgccg tcggctgtcc
     5221 atcactgtcc ttcactatgg ctttgatccc aggatgcaga tcgagaagca cctgtcggca
     5281 ccgtccgcag gggctcaaga tgcccctgtt ctcatttccg atcgcgacga tacaagtcag
     5341 gttgccagct gccgcagcag cagcagtgcc cagcaccacg agttctgcac aaggtccccc
     5401 agtaaaatga tatacattga caccagtgaa gatgcggccg tcgctagaga gagctgcgct
     5461 ggcgacgctg tagtcttcag agatggggat gctgttgatt gtagccgttg ctctttcaat
     5521 gagggtggat tcttcttgag acaaaggctt ggccatgggg ccggggttct cctccacgtc
     5581 gccggcctgc ttcagcaggc tgaagttggt ggcgccgctg cccccgggga gcatgtcaag
     5641 gtcaaaatcg tcaagagcgt cagcaggcag catatcaagg tcaaagtcgt caagggcatc
     5701 ggctgggagc atgtctaagt caaaatcgtc aagggcgtcg gtcggcccgc cgctttcgca
     5761 ctttagctgt ttctccaggc cacatatgat tagttccagg ccgaaaagga aggcaggttc
     5821 ggctccctgc cggtcgaaca gctcaattgc ttgtttcaga agtgggggca tagaatcggt
     5881 ggtaggtgtc tctctttcct cttttgctac ttgatgctcc tgttcctcca atacgcagcc
     5941 cagtgtaaag tggcccacgg cggacagagc gtacagtgcg ttctccaggg agaagccttg
     6001 ctgacacagg aacgcgagct gattttccag ggtttcgtac tgtttctctg ttgggcgggt
     6061 gccgagatgc actttagccc cgtcgcgatg tgagaggaga gcacagcggt atgacttggc
     6121 gttgttccgc agaaagtctt gccatgactc gccttccagg gggcaggagt gggtatgatg
     6181 cctgtccagc atctcgattg gcagggcatc gagcagggcc cgcttgttct tcacgtgcca
     6241 gtacagggta ggctgctcaa ctcccagctt ttgagcgagt ttccttgtcg tcaggccttc
     6301 gataccgact ccattgagta attccagagc agagtttatg actttgctct tgtccagtct
     6361 agacatctta tcgtcatcgt ctttgtaatc catggtggcg gatcccgcgt cacgacacct
     6421 gtgttctggc ggcaaacccg ttgcgaaaaa gaacgttcac ggcgactact gcacttatat
     6481 acggttctcc cccaccctcg ggaaaaaggc ggagccagta cacgacatca ctttcccagt
     6541 ttaccccgcg ccaccttctc taggcaccgg ttcaattgcc gacccctccc cccaacttct
     6601 cggggactgt gggcgatgtg cgctctgccc actgacgggc accggagcca ctcgagtgga
     6661 att
//
