LOCUS       Exported                6706 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  PiggyBac vector encoding Tet-On 3G constitutively expressed by EF1a 
            promoter.
ACCESSION   .
VERSION     .
KEYWORDS    PB-EF1a-TetOn3G
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6706)
  AUTHORS   Miller AZ, Satchie A, Tannenbaum AP, Nihal A, Thomson JA, Vereide DT
  TITLE     Expandable Arterial Endothelial Precursors from Human CD34(+) Cells 
            Differ in Their Proclivity to Undergo an Endothelial-to-Mesenchymal 
            Transition.
  JOURNAL   Stem Cell Reports. 2018 Jan 9;10(1):73-86. doi: 
            10.1016/j.stemcr.2017.12.011.
  PUBMED    29320761
REFERENCE   2  (bases 1 to 6706)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..6706
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(29..51)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     151..170
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     364..386
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     378..395
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     379..395
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     repeat_region   418..480
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        1424..2605
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          1654..2596
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     2553..2573
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     2607..2631
                     /label=LNCX
                     /note="Human CMV promoter, forward primer"
     CDS             2713..3459
                     /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"
     polyA_signal    3469..3603
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3493..3512)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3547..3566
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    4074..4107
                     /label=lox2272
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (GGATACTT). lox2272 sites are compatible with each
                     other, but incompatible with loxP or loxN sites (Lee and 
                     Saito, 1988)."
     repeat_region   4425..4459
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     primer_bind     complement(4485..4501)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4485..4501)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4498..4520)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4509..4525
                     /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(4533..4563)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4578..4599
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(4716..4733)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(4887..5475)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(4967..4986)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(5646..6506)
                     /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     6269..6288
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6507..6611)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     6679..6697
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
ORIGIN
        1 tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat gcagctcccg gagacggtca
       61 cagcttgtct gtaagcggat gccgggagca gacaagcccg tcagggcgcg tcagcgggtg
      121 ttggcgggtg tcggggctgg cttaactatg cggcatcaga gcagattgta ctgagagtgc
      181 accatatgcg gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgcc
      241 attcgccatt caggctgcgc aactgttggg aagggcgatc ggtgcgggcc tcttcgctat
      301 tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt aagttgggta acgccagggt
      361 tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt caagctttat tatttaaccc
      421 tagaaagata atcatattgt gacgtacgtt aaagataatc atgcgtaaaa ttgacgcatg
      481 tgttttatcg gtctgtatat cgaggtttat ttattaattt gaatagatat taagttttat
      541 tatatttaca cttacatact aataataaat tcaacaaaca atttatttat gtttatttat
      601 ttattaaaaa aaaacaaaaa ctcaaaattt cttctataaa gtaacaaaac ttttaaacat
      661 tctctctttt acaaaaataa acttattttg tactttaaaa acagtcatgt tgtaatcgat
      721 aataagtaat tagcttaacc tatacataat agaaacaaat tatacttatt agtcagtcag
      781 aaacaacttt ggcacatatc aatattatgc tctcgttaat taactcgagg gatccggcgc
      841 gcctgtcatt ctaaatctct ctttcagcct aaagcttttt ccccgtatcc ccccaggtgt
      901 ctgcaggctc aaagagcagc gagaagcgtt cagaggaaag cgatcccgtg ccaccttccc
      961 cgtgcccggg ctgtccccgc acgctgccgg ctcggggatg cggggggagc gccggaccgg
     1021 agcggagccc cgggcggctc gctgctgccc cctagcgggg gagggacgta attacatccc
     1081 tgggggcttt gggagggggc tgtccccgtg agctcccagg cgcgcctgtc attctaaatc
     1141 tctctttcag cctaaagctt tttccccgta tccccccagg tgtctgcagg ctcaaagagc
     1201 agcgagaagc gttcagagga aagcgatccc gtgccacctt ccccgtgccc gggctgtccc
     1261 cgcacgctgc cggctcgggg atgcgggggg agcgccggac cggagcggag ccccgggcgg
     1321 ctcgctgctg ccccctagcg ggggagggac gtaattacat ccctgggggc tttggggggg
     1381 ggctgtcccc gtgagctccc aggcgcgccg ctagcttcgt gaggctccgg tgcccgtcag
     1441 tgggcagagc gcacatcgcc cacagtcccc gagaagttgg ggggaggggt cggcaattga
     1501 accggtgcct agagaaggtg gcgcggggta aactgggaaa gtgatgtcgt gtactggctc
     1561 cgcctttttc ccgagggtgg gggagaaccg tatataagtg cagtagtcgc cgtgaacgtt
     1621 ctttttcgca acgggtttgc cgccagaaca caggtaagtg ccgtgtgtgg ttcccgcggg
     1681 cctggcctct ttacgggtta tggcccttgc gtgccttgaa ttacttccac gcccctggct
     1741 gcagtacgtg attcttgatc ccgagcttcg ggttggaagt gggtgggaga gttcgaggcc
     1801 ttgcgcttaa ggagcccctt cgcctcgtgc ttgagttgag gcctggcttg ggcgctgggg
     1861 ccgccgcgtg cgaatctggt ggcaccttcg cgcctgtctc gctgctttcg ataagtctct
     1921 agccatttaa aatttttgat gacctgctgc gacgcttttt ttctggcaag atagtcttgt
     1981 aaatgcgggc caagatctgc acactggtat ttcggttttt ggggccgcgg gcggcgacgg
     2041 ggcccgtgcg tcccagcgca catgttcggc gaggcggggc ctgcgagcgc ggccaccgag
     2101 aatcggacgg gggtagtctc aagctggccg gcctgctctg gtgcctggcc tcgcgccgcc
     2161 gtgtatcgcc ccgccctggg cggcaaggct ggcccggtcg gcaccagttg cgtgagcgga
     2221 aagatggccg cttcccggcc ctgctgcagg gagctcaaaa tggaggacgc ggcgctcggg
     2281 agagcgggcg ggtgagtcac ccacacaaag gaaaagggcc tttccgtcct cagccgtcgc
     2341 ttcatgtgac tccacggagt accgggcgcc gtccaggcac ctcgattagt tctcgagctt
     2401 ttggagtacg tcgtctttag gttgggggga ggggttttat gcgatggagt ttccccacac
     2461 tgagtgggtg gagactgaag ttaggccagc ttggcacttg atgtaattct ccttggaatt
     2521 tgcccttttt gagtttggat cttggttcat tctcaagcct cagacagtgg ttcaaagttt
     2581 ttttcttcca tttcaggtgt cgtgagagct cgtttagtga accgtcagat cgcctggaga
     2641 cgccatccac gctgttttga cctccataga agacaccggg accgatccag cctccgcggc
     2701 cccgaattca ccatgtctag actggacaag agcaaagtca taaactctgc tctggaatta
     2761 ctcaatggag tcggtatcga aggcctgacg acaaggaaac tcgctcaaaa gctgggagtt
     2821 gagcagccta ccctgtactg gcacgtgaag aacaagcggg ccctgctcga tgccctgcca
     2881 atcgagatgc tggacaggca tcatacccac tcctgccccc tggaaggcga gtcatggcaa
     2941 gactttctgc ggaacaacgc caagtcatac cgctgtgctc tcctctcaca tcgcgacggg
     3001 gctaaagtgc atctcggcac ccgcccaaca gagaaacagt acgaaaccct ggaaaatcag
     3061 ctcgcgttcc tgtgtcagca aggcttctcc ctggagaacg cactgtacgc tctgtccgcc
     3121 gtgggccact ttacactggg ctgcgtattg gaggaacagg agcatcaagt agcaaaagag
     3181 gaaagagaga cacctaccac cgattctatg cccccacttc tgaaacaagc aattgagctg
     3241 ttcgaccggc agggagccga acctgccttc cttttcggcc tggaactaat catatgtggc
     3301 ctggagaaac agctaaagtg cgaaagcggc gggccgaccg acgcccttga cgattttgac
     3361 ttagacatgc tcccagccga tgcccttgac gactttgacc ttgatatgct gcctgctgac
     3421 gctcttgacg attttgacct tgacatgctc cccgggtaac taagtaagga tccagacatg
     3481 ataagataca ttgatgagtt tggacaaacc acaactagaa tgcagtgaaa aaaatgcttt
     3541 atttgtgaaa tttgtgatgc tattgcttta tttgtaacca ttataagctg caataaacaa
     3601 gttaacaaca acaattgcat tcattttatg tttcaggttc agggggaggt gtgggaggtt
     3661 ttttaaagca agtaaaacct ctacaaatgt ggtatggctg attatgatcc ggctgcctcg
     3721 cgcttaatta acccgggggc cgcctgggaa ctgccgctgg ccccccaccg ccccaaggat
     3781 ctccggtccc cgcccggctt gctgacgtca cggatgcccc agggtgtgct gggcaggtag
     3841 cgggagcctg ggaaatggag tccattagca gaagtggcca cttccaggag tcgctgtgcc
     3901 ccgatgcaca ctgggaagtc cgcagctccg aggcccccag tggaaatcgc cagatgaggg
     3961 cctcctccgg ggaatgctgg gaaatggagt ctacaggccg gaggggtccc cacggcatac
     4021 tagcaagtgt gtgtagcacc gcgtaaaggg gatgaatagc agactgcccc gggataactt
     4081 cgtataggat actttatacg aagttatgag ctctgtgata atcactagtg aattgatact
     4141 agtcggtcac gatatctata acaagaaaat atatatataa taagttatca cgtaagtaga
     4201 acatgaaata acaatataat tatcgtatga gttaaatctt aaaagtcacg taaaagataa
     4261 tcatgcgtca ttttgactca cgcggtcgtt atagttcaaa atcagtgaca cttaccgcat
     4321 tgacaagcac gcctcacggg agctccaagc ggcgactgag atgtcctaaa tgcacagcga
     4381 cggattcgcg ctatttagaa agagagagca atatttcaag aatgcatgcg tcaattttac
     4441 gcagactatc tttctagggt taaaaaaagc ttggcgtaat catggtcata gctgtttcct
     4501 gtgtgaaatt gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt
     4561 aaagcctggg gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc
     4621 gctttccagt cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg
     4681 agaggcggtt tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
     4741 gtcgttcggc tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
     4801 gaatcagggg ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac
     4861 cgtaaaaagg ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac
     4921 aaaaatcgac gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg
     4981 tttccccctg gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac
     5041 ctgtccgcct ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat
     5101 ctcagttcgg tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
     5161 cccgaccgct gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac
     5221 ttatcgccac tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt
     5281 gctacagagt tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt
     5341 atctgcgctc tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc
     5401 aaacaaacca ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
     5461 aaaaaaggat ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
     5521 gaaaactcac gttaagggat tttggtcatg agattatcaa aaaggatctt cacctagatc
     5581 cttttaaatt aaaaatgaag ttttaaatca atctaaagta tatatgagta aacttggtct
     5641 gacagttacc aatgcttaat cagtgaggca cctatctcag cgatctgtct atttcgttca
     5701 tccatagttg cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct
     5761 ggccccagtg ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
     5821 ataaaccagc cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
     5881 atccagtcta ttaattgttg ccgggaagct agagtaagta gttcgccagt taatagtttg
     5941 cgcaacgttg ttgccattgc tacaggcatc gtggtgtcac gctcgtcgtt tggtatggct
     6001 tcattcagct ccggttccca acgatcaagg cgagttacat gatcccccat gttgtgcaaa
     6061 aaagcggtta gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta
     6121 tcactcatgg ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
     6181 ttttctgtga ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
     6241 agttgctctt gcccggcgtc aatacgggat aataccgcgc cacatagcag aactttaaaa
     6301 gtgctcatca ttggaaaacg ttcttcgggg cgaaaactct caaggatctt accgctgttg
     6361 agatccagtt cgatgtaacc cactcgtgca cccaactgat cttcagcatc ttttactttc
     6421 accagcgttt ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg
     6481 gcgacacgga aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
     6541 cagggttatt gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
     6601 ggggttccgc gcacatttcc ccgaaaagtg ccacctgacg tctaagaaac cattattatc
     6661 atgacattaa cctataaaaa taggcgtatc acgaggccct ttcgtc
//
