LOCUS       Exported                8115 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  piggyBac transposon vector that Inducibly (Tet-On) expresses Gaolf.
ACCESSION   .
VERSION     .
KEYWORDS    pB-Gaolf-Integration
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8115)
  AUTHORS   Jones EM, Jajoo R, Cancilla D, Lubock NB, Wang J, Satyadi M, Cheung 
            R, de March C, Bloom JS, Matsunami H, Kosuri S
  TITLE     A Scalable, Multiplexed Assay for Decoding GPCR-Ligand Interactions 
            with RNA Sequencing.
  JOURNAL   Cell Syst. 2019 Mar 27;8(3):254-260.e6. doi: 
            10.1016/j.cels.2019.02.009. Epub 2019 Mar 20.
  PUBMED    30904378
REFERENCE   2  (bases 1 to 8115)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8115
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        complement(1..105)
                     /gene="bla"
                     /label=AmpR promoter
     rep_origin      complement(131..586)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(268..289)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     480..499
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     713..735
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     727..744
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     728..744
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   874..936
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     protein_bind    1406..1424
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1443..1461
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1480..1498
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1517..1535
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1554..1572
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1591..1609
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1628..1646
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     protein_bind    1665..1683
                     /gene="tetO"
                     /label=tet operator
                     /bound_moiety="tetracycline repressor TetR"
                     /note="bacterial operator O2 for the tetR and tetA genes"
     primer_bind     1705..1729
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     regulatory      1819..1828
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     polyA_signal    3200..3334
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     promoter        3445..3656
                     /label=EF-1-alpha core promoter
                     /note="core promoter for human elongation factor 
                     EF-1-alpha"
     LTR             3669..3937
                     /label=5' LTR (truncated)
                     /note="truncated 5' long terminal repeat (LTR) from human 
                     T-cell leukemia virus (HTLV) type 1"
     CDS             3964..4563
                     /codon_start=1
                     /gene="pac from Streptomyces alboniger"
                     /product="puromycin N-acetyltransferase"
                     /label=PuroR
                     /note="confers resistance to puromycin"
                     /translation="MTEYKPTVRLATRDDVPRAVRTLAAAFADYPATRHTVDPDRHIER
                     VTELQELFLTRVGLDIGKVWVADDGAAVAVWTTPESVEAGAVFAEIGPRMAELSGSRLA
                     AQQQMEGLLAPHRPKEPAWFLATVGVSPDHQGKGLGSAVVLPGVEAAERAGVPAFLETS
                     APRNLPFYERLGFTVTADVEVPEGPRTWCMTRKPGA"
     primer_bind     complement(3964..3983)
                     /label=Puro-R
                     /note="Puromycin resistance gene, reverse primer. Also 
                     called puro-variant-R"
     primer_bind     4460..4480
                     /label=Puro-F
                     /note="Puromycin resistance gene, forward primer"
     primer_bind     complement(4623..4643)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     polyA_signal    4685..4806
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     repeat_region   5569..5603
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(6094..6110)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6094..6110)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6107..6129)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6118..6134
                     /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)."
     promoter        complement(6142..6172)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6187..6208
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6325..6342)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6496..7084)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6576..6595)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7255..8115)
                     /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     7878..7897
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
       61 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
      121 agtgccacct aaattgtaag cgttaatatt ttgttaaaat tcgcgttaaa tttttgttaa
      181 atcagctcat tttttaacca ataggccgaa atcggcaaaa tcccttataa atcaaaagaa
      241 tagaccgaga tagggttgag tgttgttcca gtttggaaca agagtccact attaaagaac
      301 gtggactcca acgtcaaagg gcgaaaaacc gtctatcagg gcgatggccc actacgtgaa
      361 ccatcaccct aatcaagttt tttggggtcg aggtgccgta aagcactaaa tcggaaccct
      421 aaagggagcc cccgatttag agcttgacgg ggaaagccgg cgaacgtggc gagaaaggaa
      481 gggaagaaag cgaaaggagc gggcgctagg gcgctggcaa gtgtagcggt cacgctgcgc
      541 gtaaccacca cacccgccgc gcttaatgcg ccgctacagg gcgcgtccca ttcgccattc
      601 aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg
      661 gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca
      721 cgacgttgta aaacgacggc cagtgagcgc gcctcgttca ttcacgtttt tgaacccgtg
      781 gaggacgggc agactcgcgg tgcaaatgtg ttttacagcg tgatggagca gatgaagatg
      841 ctcgacacgc tgcagaacac gcagctagat taaccctaga aagataatca tattgtgacg
      901 tacgttaaag ataatcatgt gtaaaattga cgcatgtgtt ttatcggtct gtatatcgag
      961 gtttatttat taatttgaat agatattaag ttttattata tttacactta catactaata
     1021 ataaattcaa caaacaattt atttatgttt atttatttat taaaaaaaac aaaaactcaa
     1081 aatttcttct ataaagtaac aaaactttta tgagggacag ccccccccca aagcccccag
     1141 ggatgtaatt acgtccctcc cccgctaggg ggcagcagcg agccgcccgg ggctccgctc
     1201 cggtccggcg ctccccccgc atccccgagc cggcagcgtg cggggacagc ccgggcacgg
     1261 ggaaggtggc acgggatcgc tttcctctga acgcttctcg ctgctctttg agcctgcaga
     1321 cacctggggg gatacgggga aaaggcctcc acggccacta gtccaatcat aggggtaccc
     1381 gacccatcaa gtgccacctg acgtctccct atcagtgata gagaagtcga cacgtctcga
     1441 gctccctatc agtgatagag aaggtacgtc tagaacgtct ccctatcagt gatagagaag
     1501 tcgacacgtc tcgagctccc tatcagtgat agagaaggta cgtctagaac gtctccctat
     1561 cagtgataga gaagtcgaca cgtctcgagc tccctatcag tgatagagaa ggtacgtcta
     1621 gaacgtctcc ctatcagtga tagagaagtc gacacgtctc gagctcccta tcagtgatag
     1681 agaaggtacc ccctatataa gcagagctcg tttagtgaac cgtcagatcg cctggagacg
     1741 ccatccacgc tgttttgacc tccatagaag acaccgggac cgatccagcc tgagctcgtt
     1801 tagtgaccgt ctagagccgc caccatgggg tgtttgggca acagcagcaa gaccgcggaa
     1861 gatcagggcg tagatgaaaa agaacgccgc gaggccaaca aaaagatcga gaagcagttg
     1921 cagaaagagc gcctggctta caaagcaact caccgcctgc tgcttctggg ggctggtgag
     1981 tccgggaaaa gcactatcgt caaacagatg aggatcctgc acgtcaatgg cttcaacccc
     2041 gaggaaaaga agcagaaaat tctggacatc aggaaaaatg tcaaagatgc gatcgtgaca
     2101 atcgtttcag caatgagtac tatcatacct ccagttccac tggccaaccc tgagaaccag
     2161 ttccggtcag attatatcaa gagcatagcc cctatcactg actttgaata ttcccaggag
     2221 ttctttgacc atgtgaagaa gctgtgggac gatgaaggag tgaaggcctg ctttgagaga
     2281 tccaacgagt accagctgat cgactgtgca caatacttcc tggaaaggat tgacagtgtc
     2341 agtctggttg actacacacc cacagaccag gacctgctca gatgcagagt gctgacatca
     2401 ggaatctttg agacacgatt ccaagtggac aaagtgaact ttcacatgtt tgatgttgga
     2461 ggccagagag atgagagaag aaaatggatc cagtgtttta atgatgtcac tgcgatcatt
     2521 tacgtggcgg cctgtagtag ctacaacatg gtgatccggg aagataacaa taccaacaga
     2581 cttcgggaat cactggacct gtttgaaagc atctggaata acaggtggtt gcgaaccatt
     2641 tctatcatcc tattcttgaa caaacaagac atgctggcag aaaaagtctt ggcagggaag
     2701 tcaaaaatcg aagactattt cccggagtat gccaattata ctgtccctga agatgcaaca
     2761 ccagatgcgg gagaagatcc caaagttaca agagcaaagt tctttatccg ggatctgttc
     2821 ttgaggatca gcacagccac gggtgatggc aaacattact gctaccctca cttcacctgc
     2881 gccgtggaca cagagaacat ccgcagagtg ttcaacgatt gccgtgacat catccagaga
     2941 atgcatctca agcagtacga actcttgtga gaattcccac acaggcatag agtgtctgct
     3001 attaataact atgctcaaaa attgtgtacc tttagctttt taatttgtaa aggggttaat
     3061 aaggaatatt tgatgtatag tgccttgact agagatcata atcagccata ccacatttgt
     3121 agaggtttta cttgctttaa aaaacctccc acacctcccc ctgaacctga aacataaaat
     3181 gaatgcaatt gttgttgtta acttgtttat tgcagcttat aatggttaca aataaagcaa
     3241 tagcatcaca aatttcacaa ataaagcatt tttttcactg cattctagtt gtggtttgtc
     3301 caaactcatc aatgtatctt atcatgtctg gatccccagg aagctcctct gtgtcctcat
     3361 aaaccctaac ctcctctact tgagaggaca ttccaatcat agggcggccg cgaaggatct
     3421 gcgatcgctc cggtgcccgt cagtgggcag agcgcacatc gcccacagtc cccgagaagt
     3481 tggggggagg ggtcggcaat tgaacgggtg cctagagaag gtggcgcggg gtaaactggg
     3541 aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa
     3601 gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacagctga
     3661 agcttcgagg ggctcgcatc tctccttcac gcgcccgccg ccctacctga ggccgccatc
     3721 cacgccggtt gagtcgcgtt ctgccgcctc ccgcctgtgg tgcctcctga actgcgtccg
     3781 ccgtctaggt aagtttaaag ctcaggtcga gaccgggcct ttgtccggcg ctcccttgga
     3841 gcctacctag actcagccgg ctctccacgc tttgcctgac cctgcttgct caactctacg
     3901 tctttgtttc gttttctgtt ctgcgccgtt acagatccaa gctgtgaccg gcgcctacgc
     3961 tagatgaccg agtacaagcc cacggtgcgc ctcgccaccc gcgacgacgt ccccagggcc
     4021 gtacgcaccc tcgccgccgc gttcgccgac taccccgcca cgcgccacac cgtcgatccg
     4081 gaccgccaca tcgagcgggt caccgagctg caagaactct tcctcacgcg cgtcgggctc
     4141 gacatcggca aggtgtgggt cgcggacgac ggcgccgcgg tggcggtctg gaccacgccg
     4201 gagagcgtcg aagcgggggc ggtgttcgcc gagatcggcc cgcgcatggc cgagttgagc
     4261 ggttcccggc tggccgcgca gcaacagatg gaaggcctcc tggcgccgca ccggcccaag
     4321 gagcccgcgt ggttcctggc caccgtcggc gtctcgcccg accaccaggg caagggtctg
     4381 ggcagcgccg tcgtgctccc cggagtggag gcggccgagc gcgccggggt gcccgccttc
     4441 ctggagacct ccgcgccccg caacctcccc ttctacgagc ggctcggctt caccgtcacc
     4501 gccgacgtcg aggtgcccga aggaccgcgc acctggtgca tgacccgcaa gcccggtgcc
     4561 tgagtcgaca atcaacctct ggattacaaa atttgtgaaa gattgactgg tattcttaac
     4621 tatgttgctc cttttacgct atgtggatac gctgctttaa tgcctttgta tcatgcgtta
     4681 actaaacttg tttattgcag cttataatgg ttacaaataa agcaatagca tcacaaattt
     4741 cacaaataaa gcattttttt cactgcattc tagttgtggt ttgtccaaac tcatcaatgt
     4801 atcttatcat gtctggaatt gactcaaatg atgtcaatta gtctatcaga agctcatctg
     4861 gtctcccttc cgggggacaa gacatccctg tttaatattt aaacagcagt gttcccaaac
     4921 tgggttctta tatcccttgc tctggtcaac caggttgcag ggtttcctgt cctcacagga
     4981 acgaagtccc taaagaaaca gtggcagcca ggtttagccc cggaattgac tggattcctt
     5041 ttttagggcc cattggtatg gctttttccc cgtatccccc caggtgtctg caggctcaaa
     5101 gagcagcgag aagcgttcag aggaaagcga tcccgtgcca ccttccccgt gcccgggctg
     5161 tccccgcacg ctgccggctc ggggatgcgg ggggagcgcc ggaccggagc ggagccccgg
     5221 gcggctcgct gctgccccct agcgggggag ggacgtaatt acatccctgg gggctttggg
     5281 ggggggctgt ccctgatatc tataacaaga aaatatatat ataataagtt atcacgtaag
     5341 tagaacatga aataacaata taattatcgt atgagttaaa tcttaaaagt cacgtaaaag
     5401 ataatcatgc gtcattttga ctcacgcggt cgttatagtt caaaatcagt gacacttacc
     5461 gcattgacaa gcacgcctca cgggagctcc aagcggcgac tgagatgtcc taaatgcaca
     5521 gcgacggatt cgcgctattt agaaagagag agcaatattt caagaatgca tgcgtcaatt
     5581 ttacgcagac tatctttcta gggttaatct agctgcatca ggatcatatc gtcgggtctt
     5641 ttttccggct cagtcatcgc ccaagctggc gctatctggg catcggggag gaagaagccc
     5701 gtgccttttc ccgcgaggtt gaagcggcat ggaaagagtt tgccgaggat gactgctgct
     5761 gcattgacgt tgagcgaaaa cgcacgttta ccatgatgat tcgggaaggt gtggccatgc
     5821 acgcctttaa cggtgaactg ttcgttcagg ccacctggga taccagttcg tcgcggcttt
     5881 tccggacaca gttccggatg gtcagcccga agcgcatcag caacccgaac aataccggcg
     5941 acagccggaa ctgccgtgcc ggtgtgcaga ttaatgacag cggtgcggcg ctgggatatt
     6001 acgtcagcga ggacgggtat cctggctgga tgccgcagaa atggacatgg ataccccgtg
     6061 agttacccgg cgggcgcgct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg
     6121 ttatccgctc acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg
     6181 tgcctaatga gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc
     6241 gggaaacctg tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt
     6301 gcgtattggg cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct
     6361 gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga
     6421 taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc
     6481 cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg
     6541 ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg
     6601 aagctccctc gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt
     6661 tctcccttcg ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt
     6721 gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg
     6781 cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact
     6841 ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt
     6901 cttgaagtgg tggcctaact acggctacac tagaagaaca gtatttggta tctgcgctct
     6961 gctgaagcca gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac
     7021 cgctggtagc ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc
     7081 tcaagaagat cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg
     7141 ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta
     7201 aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca
     7261 atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc
     7321 ctgactcccc gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc
     7381 tgcaatgata ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc
     7441 agccggaagg gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat
     7501 taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt
     7561 tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc
     7621 cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag
     7681 ctccttcggt cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt
     7741 tatggcagca ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac
     7801 tggtgagtac tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg
     7861 cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat
     7921 tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc
     7981 gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc
     8041 tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa
     8101 atgttgaata ctcat
//
