LOCUS       Exported                7873 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Mammalian expression vector for tetracycline-transactivator (rtTA), 
            IRES-Hygro.
ACCESSION   .
VERSION     .
KEYWORDS    pPB-CAG-rtTA-IRES-Hygro
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7873)
  AUTHORS   Khoueiry R, Sohni A, Thienpont B, Luo X, Velde JV, Bartoccetti M, 
            Boeckx B, Zwijsen A, Rao A, Lambrechts D, Koh KP
  TITLE     Lineage-specific functions of TET1 in the postimplantation mouse 
            embryo.
  JOURNAL   Nat Genet. 2017 Jul;49(7):1061-1072. doi: 10.1038/ng.3868. Epub 2017
            May 15.
  PUBMED    28504700
REFERENCE   2  (bases 1 to 7873)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7873
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      complement(3..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     623..642
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        623..641
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     repeat_region   660..694
                     /label=piggyBac left (5') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     enhancer        1052..1355
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1360..1637
                     /label=chicken beta-actin promoter
     intron          1638..2654
                     /label=chimeric intron
                     /note="chimera between introns from chicken beta-actin and 
                     rabbit beta-globin"
     primer_bind     2578..2600
                     /label=pCAGGS-5
                     /note="Chimeric intron in CAG promoter, forward primer"
     primer_bind     2662..2681
                     /label=pCAG-F
                     /note="Rabbit beta-globin intron, for pCAG plasmids, 
                     forward primer"
     CDS             2721..3467
                     /codon_start=1
                     /product="improved tetracycline-controlled transactivator"
                     /label=rtTA-Advanced
                     /note="In the Tet-On(R) system, rtTA-Advanced binds to the 
                     Tet-responsive element and stimulates transcription only in
                     the presence of tetracycline or doxycycline."
                     /translation="MSRLDKSKVINGALELLNGVGIEGLTTRKLAQKLGVEQPTLYWHV
                     KNKRALLDALPIEMLDRHHTHFCPLEGESWQDFLRNNAKSFRCALLSHRDGAKVHLGTR
                     PTEKQYETLENQLAFLCQQGFSLENALYALSAVGHFTLGCVLEEQEHQVAKEERETPTT
                     DSMPPLLRQAIELFDRQGAEPAFLFGLELIICGLEKQLKCESGGPADALDDFDLDMLPA
                     DALDDFDLDMLPADALDDFDLDMLPG"
     misc_feature    3503..4076
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3670..3687)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     3897..3916
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4115..5134
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGYV
                     LRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPET
                     ELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQTV
                     MDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFGD
                     SQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGNF
                     DDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE"
     primer_bind     5239..5258
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    5376..5424
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
     repeat_region   5599..5661
                     /label=piggyBac right (3') inverted repeat
                     /note="piggyBac transposon-specific inverted terminal 
                     repeat sequence (ITR)"
     promoter        complement(5685..5703)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5724..5740)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5724..5740)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(5737..5759)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    5748..5764
                     /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(5772..5802)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5817..5838
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5955..5972)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(6126..6714)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(6206..6225)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(6885..7745)
                     /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     7508..7527
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(7746..7850)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgaat tgtaatacga ctcactatag ggcgaattgg gtaccttaac
      661 cctagaaaga tagtctgcgt aaaattgacg catgcattct tgaaatattg ctctctcttt
      721 ctaaatagcg cgaatccgtc gctgtgcatt taggacatct cagtcgccgc ttggagctcc
      781 cgtgaggcgt gcttgtcaat gcggtaagtg tcactgattt tgaactataa cgaccgcgtg
      841 agtcaaaatg acgcatgatt atcttttacg tgacttttaa gatttaactc atacgataat
      901 tatattgtta tttcatgttc tacttacgtg ataacttatt atatatatat tttcttgtta
      961 tagatatcgt cgacattgat tattgactag ctagttatta atagtaatca attacggggt
     1021 cattagttca tagcccatat atggagttcc gcgttacata acttacggta aatggcccgc
     1081 ctggctgacc gcccaacgac ccccgcccat tgacgtcaat aatgacgtat gttcccatag
     1141 taacgccaat agggactttc cattgacgtc aatgggtgga gtatttacgg taaactgccc
     1201 acttggcagt acatcaagtg tatcatatgc caagtacgcc ccctattgac gtcaatgacg
     1261 gtaaatggcc cgcctggcat tatgcccagt acatgacctt atgggacttt cctacttggc
     1321 agtacatcta cgtattagtc atcgctatta ccatgatggt cgaggtgagc cccacgttct
     1381 gcttcactct ccccatctcc cccccctccc cacccccaat tttgtattta tttatttttt
     1441 aattattttg tgcagcgatg ggggcggggg gggggggggg gcgcgcgcca ggcggggcgg
     1501 ggcggggcga ggggcggggc ggggcgaggc ggagaggtgc ggcggcagcc aatcagagcg
     1561 gcgcgctccg aaagtttcct tttatggcga ggcggcggcg gcggcggccc tataaaaagc
     1621 gaagcgcgcg gcgggcggga gtcgctgcgc gctgccttcg ccccgtgccc cgctccgccg
     1681 ccgcctcgcg ccgcccgccc cggctctgac tgaccgcgtt actcccacag gtgagcgggc
     1741 gggacggccc ttctcctccg ggctgtaatt agcgcttggt ttaatgacgg cttgtttctt
     1801 ttctgtggct gcgtgaaagc cttgaggggc tccgggaggg ccctttgtgc ggggggagcg
     1861 gctcgggggg tgcgtgcgtg tgtgtgtgcg tggggagcgc cgcgtgcggc tccgcgctgc
     1921 ccggcggctg tgagcgctgc gggcgcggcg cggggctttg tgcgctccgc agtgtgcgcg
     1981 aggggagcgc ggccgggggc ggtgccccgc ggtgcggggg gggctgcgag gggaacaaag
     2041 gctgcgtgcg gggtgtgtgc gtgggggggt gagcaggggg tgtgggcgcg tcggtcgggc
     2101 tgcaaccccc cctgcacccc cctccccgag ttgctgagca cggcccggct tcgggtgcgg
     2161 ggctccgtac ggggcgtggc gcggggctcg ccgtgccggg cggggggtgg cggcaggtgg
     2221 gggtgccggg cggggcgggg ccgcctcggg ccggggaggg ctcgggggag gggcgcggcg
     2281 gcccccggag cgccggcggc tgtcgaggcg cggcgagccg cagccattgc cttttatggt
     2341 aatcgtgcga gagggcgcag ggacttcctt tgtcccaaat ctgtgcggag ccgaaatctg
     2401 ggaggcgccg ccgcaccccc tctagcgggc gcggggcgaa gcggtgcggc gccggcagga
     2461 aggaaatggg cggggagggc cttcgtgcgt cgccgcgccg ccgtcccctt ctccctctcc
     2521 agcctcgggg ctgtccgcgg ggggacggct gccttcgggg gggacggggc agggcggggt
     2581 tcggcttctg gcgtgtgacc ggcggctcta gagcctctgc taaccatgtt catgccttct
     2641 tctttttcct acagctcctg ggcaacgtgc tggttattgt gctgtctcat cattttggca
     2701 aagaattcct cgaggccacc atgtctagac tggacaagag caaagtcata aacggcgctc
     2761 tggaattact caatggagtc ggtatcgaag gcctgacgac aaggaaactc gctcaaaagc
     2821 tgggagttga gcagcctacc ctgtactggc acgtgaagaa caagcgggcc ctgctcgatg
     2881 ccctgccaat cgagatgctg gacaggcatc atacccactt ctgccccctg gaaggcgagt
     2941 catggcaaga ctttctgcgg aacaacgcca agtcattccg ctgtgctctc ctctcacatc
     3001 gcgacggggc taaagtgcat ctcggcaccc gcccaacaga gaaacagtac gaaaccctgg
     3061 aaaatcagct cgcgttcctg tgtcagcaag gcttctccct ggagaacgca ctgtacgctc
     3121 tgtccgccgt gggccacttt acactgggct gcgtattgga ggaacaggag catcaagtag
     3181 caaaagagga aagagagaca cctaccaccg attctatgcc cccacttctg agacaagcaa
     3241 ttgagctgtt cgaccggcag ggagccgaac ctgccttcct tttcggcctg gaactaatca
     3301 tatgtggcct ggagaaacag ctaaagtgcg aaagcggcgg gccggccgac gcccttgacg
     3361 attttgactt agacatgctc ccagccgatg cccttgacga ctttgacctt gatatgctgc
     3421 ctgctgacgc tcttgacgat tttgaccttg acatgctccc cgggtaagct cgagactagt
     3481 tctagagcgg ccgcggatcc cgcccctctc cctccccccc ccctaacgtt actggccgaa
     3541 gccgcttgga ataaggccgg tgtgcgtttg tctatatgtt attttccacc atattgccgt
     3601 cttttggcaa tgtgagggcc cggaaacctg gccctgtctt cttgacgagc attcctaggg
     3661 gtctttcccc tctcgccaaa ggaatgcaag gtctgttgaa tgtcgtgaag gaagcagttc
     3721 ctctggaagc ttcttgaaga caaacaacgt ctgtagcgac cctttgcagg cagcggaacc
     3781 ccccacctgg cgacaggtgc ctctgcggcc aaaagccacg tgtataagat acacctgcaa
     3841 aggcggcaca accccagtgc cacgttgtga gttggatagt tgtggaaaga gtcaaatggc
     3901 tctcctcaag cgtattcaac aaggggctga aggatgccca gaaggtaccc cattgtatgg
     3961 gatctgatct ggggcctcgg tgcacatgct ttacatgtgt ttagtcgagg ttaaaaaaac
     4021 gtctaggccc cccgaaccac ggggacgtgg ttttcctttg aaaaacacga tgataagctt
     4081 gccacaaccc gtaccaaaga tggatagatc cggaaagcct gaactcaccg cgacgtctgt
     4141 cgagaagttt ctgatcgaaa agttcgacag cgtctccgac ctgatgcagc tctcggaggg
     4201 cgaagaatct cgtgctttca gcttcgatgt aggagggcgt ggatatgtcc tgcgggtaaa
     4261 tagctgcgcc gatggtttct acaaagatcg ttatgtttat cggcactttg catcggccgc
     4321 gctcccgatt ccggaagtgc ttgacattgg ggaattcagc gagagcctga cctattgcat
     4381 ctcccgccgt gcacagggtg tcacgttgca agacctgcct gaaaccgaac tgcccgctgt
     4441 tctgcagccg gtcgcggagg ccatggatgc gatcgctgcg gccgatctta gccagacgag
     4501 cgggttcggc ccattcggac cgcaaggaat cggtcaatac actacatggc gtgatttcat
     4561 atgcgcgatt gctgatcccc atgtgtatca ctggcaaact gtgatggacg acaccgtcag
     4621 tgcgtccgtc gcgcaggctc tcgatgagct gatgctttgg gccgaggact gccccgaagt
     4681 ccggcacctc gtgcacgcgg atttcggctc caacaatgtc ctgacggaca atggccgcat
     4741 aacagcggtc attgactgga gcgaggcgat gttcggggat tcccaatacg aggtcgccaa
     4801 catcttcttc tggaggccgt ggttggcttg tatggagcag cagacgcgct acttcgagcg
     4861 gaggcatccg gagcttgcag gatcgccgcg gctccgggcg tatatgctcc gcattggtct
     4921 tgaccaactc tatcagagct tggttgacgg caatttcgat gatgcagctt gggcgcaggg
     4981 tcgatgcgac gcaatcgtcc gatccggagc cgggactgtc gggcgtacac aaatcgcccg
     5041 cagaagcgcg gccgtctgga ccgatggctg tgtagaagta ctcgccgata gtggaaaccg
     5101 acgccccagc actcgtccga gggcaaagga atagccatgg cgatgcctgc ttgccgaata
     5161 tcatggtgga aaatggccgc ttttctggat tcatcgactg tggccggctg ggtgtggcgg
     5221 accgctatca ggacatagcg ttggctaccc gtgatattgc tgaagagctt ggcggcgaat
     5281 gggctgaccg cttcctcgtg ctttacggta tcgccgctcc cgattcgcag cgcatcgcct
     5341 tctatcgcct tcttgacgag ttcttctgag gggatcggca ataaaaagac agaataaaac
     5401 gcacgggtgt tgggtcgttt gttcggatcc tttgttactt tatagaagaa attttgagtt
     5461 tttgtttttt tttaataaat aaataaacat aaataaattg tttgttgaat ttattattag
     5521 tatgtaagtg taaatataat aaaacttaat atctattcaa attaataaat aaacctcgat
     5581 atacagaccg ataaaacaca tgcgtcaatt ttacgcatga ttatctttaa cgtacgtcac
     5641 aatatgatta tctttctagg gttaaagagc tccagctttt gttcccttta gtgagggtta
     5701 atttcgagct tggcgtaatc atggtcatag ctgtttcctg tgtgaaattg ttatccgctc
     5761 acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga
     5821 gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg
     5881 tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg
     5941 cgctcttccg cttcctcgct cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg
     6001 gtatcagctc actcaaaggc ggtaatacgg ttatccacag aatcagggga taacgcagga
     6061 aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg
     6121 gcgtttttcc ataggctccg cccccctgac gagcatcaca aaaatcgacg ctcaagtcag
     6181 aggtggcgaa acccgacagg actataaaga taccaggcgt ttccccctgg aagctccctc
     6241 gtgcgctctc ctgttccgac cctgccgctt accggatacc tgtccgcctt tctcccttcg
     6301 ggaagcgtgg cgctttctca tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt
     6361 cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc
     6421 ggtaactatc gtcttgagtc caacccggta agacacgact tatcgccact ggcagcagcc
     6481 actggtaaca ggattagcag agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg
     6541 tggcctaact acggctacac tagaaggaca gtatttggta tctgcgctct gctgaagcca
     6601 gttaccttcg gaaaaagagt tggtagctct tgatccggca aacaaaccac cgctggtagc
     6661 ggtggttttt ttgtttgcaa gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat
     6721 cctttgatct tttctacggg gtctgacgct cagtggaacg aaaactcacg ttaagggatt
     6781 ttggtcatga gattatcaaa aaggatcttc acctagatcc ttttaaatta aaaatgaagt
     6841 tttaaatcaa tctaaagtat atatgagtaa acttggtctg acagttacca atgcttaatc
     6901 agtgaggcac ctatctcagc gatctgtcta tttcgttcat ccatagttgc ctgactcccc
     6961 gtcgtgtaga taactacgat acgggagggc ttaccatctg gccccagtgc tgcaatgata
     7021 ccgcgagacc cacgctcacc ggctccagat ttatcagcaa taaaccagcc agccggaagg
     7081 gccgagcgca gaagtggtcc tgcaacttta tccgcctcca tccagtctat taattgttgc
     7141 cgggaagcta gagtaagtag ttcgccagtt aatagtttgc gcaacgttgt tgccattgct
     7201 acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt cattcagctc cggttcccaa
     7261 cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa aagcggttag ctccttcggt
     7321 cctccgatcg ttgtcagaag taagttggcc gcagtgttat cactcatggt tatggcagca
     7381 ctgcataatt ctcttactgt catgccatcc gtaagatgct tttctgtgac tggtgagtac
     7441 tcaaccaagt cattctgaga atagtgtatg cggcgaccga gttgctcttg cccggcgtca
     7501 atacgggata ataccgcgcc acatagcaga actttaaaag tgctcatcat tggaaaacgt
     7561 tcttcggggc gaaaactctc aaggatctta ccgctgttga gatccagttc gatgtaaccc
     7621 actcgtgcac ccaactgatc ttcagcatct tttactttca ccagcgtttc tgggtgagca
     7681 aaaacaggaa ggcaaaatgc cgcaaaaaag ggaataaggg cgacacggaa atgttgaata
     7741 ctcatactct tcctttttca atattattga agcatttatc agggttattg tctcatgagc
     7801 ggatacatat ttgaatgtat ttagaaaaat aaacaaatag gggttccgcg cacatttccc
     7861 cgaaaagtgc cac
//
