LOCUS       sequence-467765-        6511 bp    DNA     circular SYN 15-APR-2025
DEFINITION  Cre expression under the control of HSP16F promoter; Tet-On 
            3G-inducible PiggyBac system for expression in human pluripotent 
            stem cells..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6511)
  TITLE     Heat shock promoters in human pluripotent stem cells
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 6511)
  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            "Unpublished"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: PB-HSP16F-Cre
FEATURES             Location/Qualifiers
     source          1..6511
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     regulatory      2..11
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             14..34
                     /codon_start=1
                     /product="nuclear localization signal of SV40 (simian virus
                     40) large T antigen"
                     /label=SV40 NLS
                     /translation="PKKKRKV"
     CDS             35..1063
                     /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    1244..1378
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   1654..1688
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(1699..1716)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(1699..1715)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(1708..1730)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     complement(1924..1943)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     2043..2065
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(2103..2121)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2189..2293
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2294..2362,2363..3154)
                     /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(2512..2531)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3325..3913
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3814..3833
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4067..4084
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    4201..4222
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(4237..4242,4243..4260,4261..4267)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4275..4291
                     /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     4280..4302
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     4299..4315
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4299..4315
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     repeat_region   4341..4403
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     polyA_signal    4579..4660
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     CDS             complement(4777..5175)
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     CDS             complement(5176..5232)
                     /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(5242..5985)
                     /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(5986..6009)
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     regulatory      6009..6018
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     promoter        complement(6038..6249)
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
ORIGIN
        1 cgccaccatg ggcccaaaga agaagagaaa ggtttcgaat ttactgaccg tacaccaaaa
       61 tttgcctgca ttaccggtcg atgcaacgag tgatgaggtt cgcaagaacc tgatggacat
      121 gttcagggat cgccaggcgt tttctgagca tacctggaaa atgcttctgt ccgtttgccg
      181 gtcgtgggcg gcatggtgca agttgaataa ccggaaatgg tttcccgcag aacctgaaga
      241 tgttcgcgat tatcttctat atcttcaggc gcgcggtctg gcagtaaaaa ctatccagca
      301 acatttgggc cagctaaaca tgcttcatcg tcggtccggg ctgccacgac caagtgacag
      361 caatgctgtt tcactggtta tgcggcggat ccgaaaagaa aacgttgatg ccggtgaacg
      421 tgcaaaacag gctctagcgt tcgaacgcac tgatttcgac caggttcgtt cactcatgga
      481 aaatagcgat cgctgccagg atatacgtaa tctggcattt ctggggattg cttataacac
      541 cctgttacgt atagccgaaa ttgccaggat cagggttaaa gatatctcac gtactgacgg
      601 tgggagaatg ttaatccata ttggcagaac gaaaacgctg gttagcaccg caggtgtaga
      661 gaaggcactt agcctggggg taactaaact ggtcgagcga tggatttccg tctctggtgt
      721 agctgatgat ccgaataact acctgttttg ccgggtcaga aaaaatggtg ttgccgcgcc
      781 atctgccacc agccagctat caactcgcgc cctggaaggg atttttgaag caactcatcg
      841 attgatttac ggcgctaagg atgactctgg tcagagatac ctggcctggt ctggacacag
      901 tgcccgtgtc ggagccgcgc gagatatggc ccgcgctgga gtttcaatac cggagatcat
      961 gcaagctggt ggctggacca atgtaaatat tgtcatgaac tatatccgta acctggatag
     1021 tgaaacaggg gcaatggtgc gcctgctgga agatggcgat tagactagta gaccacctcc
     1081 cctgcgagct aagctggaca gccaatgacg ggtaagagag tgacattttt cactaaccta
     1141 agacaggagg gccgtcagag ctactgccta atccaaagac gggtaaaagt gataaaaatg
     1201 tatcactcca acctaagaca ggcgcagctt ccgagggatt tgagatccag acatgataag
     1261 atacattgat gagtttggac aaaccaaaac tagaatgcag tgaaaaaaat gccttatttg
     1321 tgaaatttgt gatgctattg ccttatttgt aaccattata agctgcaata aacaagtttg
     1381 atatctataa caagaaaata tatatataat aagttatcac gtaagtagaa catgaaataa
     1441 caatataatt atcgtatgag ttaaatctta aaagtcacgt aaaagataat catgcgtcat
     1501 tttgactcac gcggtcgtta tagttcaaaa tcagtgacac ttaccgcatt gacaagcacg
     1561 cctcacggga gctccaagcg gcgactgaga tgtcctaaat gcacagcgac ggattcgcgc
     1621 tatttagaaa gagagagcaa tatttcaaga atgcatgcgt caattttacg cagactatct
     1681 ttctagggtt aagaattcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg
     1741 cgttacccaa cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga
     1801 agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatggcgcct
     1861 gatgcggtat tttctcctta cgcatctgtg cggtatttca caccgcatat ggtgcactct
     1921 cagtacaatc tgctctgatg ccgcatagtt aagccagccc cgacacccgc caacacccgc
     1981 tgacgcgccc tgacgggctt gtctgctccc ggcatccgct tacagacaag ctgtgaccgt
     2041 ctccgggagc tgcatgtgtc agaggttttc accgtcatca ccgaaacgcg cgagacgaaa
     2101 gggcctcgtg atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac
     2161 gtcaggtggc acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat
     2221 acattcaaat atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg
     2281 aaaaaggaag agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc
     2341 attttgcctt cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga
     2401 tcagttgggt gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga
     2461 gagttttcgc cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg
     2521 cgcggtatta tcccgtattg acgccgggca agagcaactc ggtcgccgca tacactattc
     2581 tcagaatgac ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac
     2641 agtaagagaa ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact
     2701 tctgacaacg atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca
     2761 tgtaactcgc cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg
     2821 tgacaccacg atgcctgtag caatggcaac aacgttgcgc aaactattaa ctggcgaact
     2881 acttactcta gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg
     2941 accacttctg cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg
     3001 tgagcgtggg tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat
     3061 cgtagttatc tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc
     3121 tgagataggt gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat
     3181 actttagatt gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt
     3241 tgataatctc atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc
     3301 cgtagaaaag atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt
     3361 gcaaacaaaa aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac
     3421 tctttttccg aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt
     3481 gtagccgtag ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct
     3541 gctaatcctg ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga
     3601 ctcaagacga tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac
     3661 acagcccagc ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg
     3721 agaaagcgcc acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt
     3781 cggaacagga gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc
     3841 tgtcgggttt cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg
     3901 gagcctatgg aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc
     3961 ttttgctcac atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc
     4021 ctttgagtga gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag
     4081 cgaggaagcg gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca
     4141 ttaatgcagc tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
     4201 taatgtgagt tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
     4261 tatgttgtgt ggaattgtga gcggataaca atttcacaca ggaaacagct atgaccatga
     4321 ttacgccaag gtcgacttaa ccctagaaag ataatcatat tgtgacgtac gttaaagata
     4381 atcatgcgta aaattgacgc atgtgtttta tcggtctgta tatcgaggtt tatttattaa
     4441 tttgaataga tattaagttt tattatattt acacttacat actaataata aattcaacaa
     4501 acaatttatt tatgtttatt tatttattaa aaaaaaacaa aaactcaaaa tttcttctat
     4561 aaagtaacaa aacttttagc agtgaaaaaa atgctttatt tgtgaaattt gtgatgctat
     4621 tgctttattt gtaaccatta taagctgcaa taaacaagtt aacaacaaca attgcattca
     4681 ttttatgttt caggttcagg gggaggtgtg ggaggttttt taaagcaagt aaaacctcta
     4741 caaatgtggt atggctgatt atgatcctct ggagatttag ccctcccaca cataaccaga
     4801 gggcagcaat tcacgaatcc caactgccgt cggctgtcca tcactgtcct tcactatggc
     4861 tttgatccca ggatgcagat cgagaagcac ctgtcggcac cgtccgcagg ggctcaagat
     4921 gcccctgttc tcatttccga tcgcgacgat acaagtcagg ttgccagctg ccgcagcagc
     4981 agcagtgccc agcaccacga gttctgcaca aggtccccca gtaaaatgat atacattgac
     5041 accagtgaag atgcggccgt cgctagagag agctgcgctg gcgacgctgt agtcttcaga
     5101 gatggggatg ctgttgattg tagccgttgc tctttcaatg agggtggatt cttcttgaga
     5161 caaaggcttg gccatggggc cggggttctc ctccacgtcg ccggcctgct tcagcaggct
     5221 gaagttggtg gcgccgctgc ccccggggag catgtcaagg tcaaaatcgt caagagcgtc
     5281 agcaggcagc atatcaaggt caaagtcgtc aagggcatcg gctgggagca tgtctaagtc
     5341 aaaatcgtca agggcgtcgg tcggcccgcc gctttcgcac tttagctgtt tctccaggcc
     5401 acatatgatt agttccaggc cgaaaaggaa ggcaggttcg gctccctgcc ggtcgaacag
     5461 ctcaattgct tgtttcagaa gtgggggcat agaatcggtg gtaggtgtct ctctttcctc
     5521 ttttgctact tgatgctcct gttcctccaa tacgcagccc agtgtaaagt ggcccacggc
     5581 ggacagagcg tacagtgcgt tctccaggga gaagccttgc tgacacagga acgcgagctg
     5641 attttccagg gtttcgtact gtttctctgt tgggcgggtg ccgagatgca ctttagcccc
     5701 gtcgcgatgt gagaggagag cacagcggta tgacttggcg ttgttccgca gaaagtcttg
     5761 ccatgactcg ccttccaggg ggcaggagtg ggtatgatgc ctgtccagca tctcgattgg
     5821 cagggcatcg agcagggccc gcttgttctt cacgtgccag tacagggtag gctgctcaac
     5881 tcccagcttt tgagcgagtt tccttgtcgt caggccttcg ataccgactc cattgagtaa
     5941 ttccagagca gagtttatga ctttgctctt gtccagtcta gacatcttat cgtcatcgtc
     6001 tttgtaatcc atggtggcgg atcccgcgtc acgacacctg tgttctggcg gcaaacccgt
     6061 tgcgaaaaag aacgttcacg gcgactactg cacttatata cggttctccc ccaccctcgg
     6121 gaaaaaggcg gagccagtac acgacatcac tttcccagtt taccccgcgc caccttctct
     6181 aggcaccggt tcaattgccg acccctcccc ccaacttctc ggggactgtg ggcgatgtgc
     6241 gctctgccca ctgacgggca ccggagccac tcgagtggga ttgtagtttg aagatttcac
     6301 aattagagtg aatgttgttt ggttcggttt tgtcactgta tttatactca tttccacctt
     6361 tttctagaag gtcctagatg catctaggac cttctagaac attctaaacg gctgcaggat
     6421 acgggtatat aagccaatcg tgttcagagg aaaccaatac actttgttca agtgcttact
     6481 gttcattctc taaacttcaa gacaccggta c
//
