LOCUS       Exported                8857 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expression of mutated catalytic domain only form of TET1.
ACCESSION   .
VERSION     .
KEYWORDS    pEF1a_CD MUT TET1
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 8857)
  AUTHORS   Vincenzetti L, Leoni C, Chirichella M, Kwee I, Monticelli S
  TITLE     The contribution of active and passive mechanisms of 5mC and 5hmC 
            removal in human T lymphocytes is differentiation- and 
            activation-dependent.
  JOURNAL   Eur J Immunol. 2019 Jan 30. doi: 10.1002/eji.201847967.
  PUBMED    30698829
REFERENCE   2  (bases 1 to 8857)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..8857
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     primer_bind     complement(45..64)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     promoter        352..1530
                     /label=EF-1-alpha promoter
                     /note="strong constitutive promoter for human elongation 
                     factor EF-1-alpha"
     intron          583..1521
                     /label=EF-1-alpha intron A
                     /note="intron upstream of the start codon of human 
                     EF-1-alpha"
     primer_bind     1478..1498
                     /label=EF1a-F
                     /note="Human elongation factor-1a promoter, forward primer"
     primer_bind     1547..1566
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1547..1565
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     regulatory      1598..1607
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     CDS             1607..1630
                     /codon_start=1
                     /product="FLAG(R) epitope tag, followed by an enterokinase 
                     cleavage site"
                     /label=FLAG
                     /translation="DYKDDDDK"
     CDS             1643..1669
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     CDS             4367..4408
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             4418..4435
                     /codon_start=1
                     /product="6xHis affinity tag"
                     /label=6xHis
                     /translation="HHHHHH"
     primer_bind     complement(4458..4475)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    4464..4688
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      4734..5162
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(4821..4840)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     5031..5052
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(5171..5191)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     promoter        5176..5505
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     rep_origin      5356..5491
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     5418..5437
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             5572..6366
                     /codon_start=1
                     /gene="aph(3')-II (or nptII)"
                     /product="aminoglycoside phosphotransferase from Tn5"
                     /label=NeoR/KanR
                     /note="confers resistance to neomycin, kanamycin, and G418 
                     (Geneticin(R))"
                     /translation="MIEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRP
                     VLFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLS
                     SHLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQ
                     GLAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(5626..5645)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     6236..6255
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     polyA_signal    6542..6663
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(6579..6598)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     6633..6652
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(6712..6728)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(6712..6728)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(6725..6747)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    6736..6752
                     /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(6760..6790)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    6805..6826
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(6943..6960)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(7103..7691)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(7183..7202)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(7862..8722)
                     /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     8485..8504
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(8723..8827)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 cgacggatcg ggagatctcc cgatccccta tggtgcactc tcagtacaat ctgctctgat
       61 gccgcatagt taagccagta tctgctccct gcttgtgtgt tggaggtcgc tgagtagtgc
      121 gcgagcaaaa tttaagctac aacaaggcaa ggcttgaccg acaattgcat gaagaatctg
      181 cttagggtta ggcgttttgc gctgcttcgc gatgtacggg ccagatatac gcgttgacat
      241 tgattattga ctaggctttt gcaaaaagct ttgcaaagat ggataaagtt ttaaacagag
      301 aggaatcttt gcagctaatg gaccttctag gtcttgaaag gagtgggaat tggctccggt
      361 gcccgtcagt gggcagagcg cacatcgccc acagtccccg agaagttggg gggaggggtc
      421 ggcaattgaa ccggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg
      481 tactggctcc gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc
      541 gtgaacgttc tttttcgcaa cgggtttgcc gccagaacac aggtaagtgc cgtgtgtggt
      601 tcccgcgggc ctggcctctt tacgggttat ggcccttgcg tgccttgaat tacttccacc
      661 tggctgcagt acgtgattct tgatcccgag cttcgggttg gaagtgggtg ggagagttcg
      721 aggccttgcg cttaaggagc cccttcgcct cgtgcttgag ttgaggcctg gcctgggcgc
      781 tggggccgcc gcgtgcgaat ctggtggcac cttcgcgcct gtctcgctgc tttcgataag
      841 tctctagcca tttaaaattt ttgatgacct gctgcgacgc tttttttctg gcaagatagt
      901 cttgtaaatg cgggccaaga tctgcacact ggtatttcgg tttttggggc cgcgggcggc
      961 gacggggccc gtgcgtccca gcgcacatgt tcggcgaggc ggggcctgcg agcgcggcca
     1021 ccgagaatcg gacgggggta gtctcaagct ggccggcctg ctctggtgcc tggcctcgcg
     1081 ccgccgtgta tcgccccgcc ctgggcggca aggctggccc ggtcggcacc agttgcgtga
     1141 gcggaaagat ggccgcttcc cggccctgct gcagggagct caaaatggag gacgcggcgc
     1201 tcgggagagc gggcgggtga gtcacccaca caaaggaaaa gggcctttcc gtcctcagcc
     1261 gtcgcttcat gtgactccac ggagtaccgg gcgccgtcca ggcacctcga ttagttctcg
     1321 agcttttgga gtacgtcgtc tttaggttgg ggggaggggt tttatgcgat ggagtttccc
     1381 cacactgagt gggtggagac tgaagttagg ccagcttggc acttgatgta attctccttg
     1441 gaatttgccc tttttgagtt tggatcttgg ttcattctca agcctcagac agtggttcaa
     1501 agtttttttc ttccatttca ggtgtcgtga ggaattagct tggtactaat acgactcact
     1561 atagggagac ccaagctggc taggtaagct tggtaccgcc accatggact acaaggacga
     1621 cgatgacaag ctcgatggag gataccccta cgacgtgccc gactacgccg gaggatatcc
     1681 tgaatcagca gaggaaaagg tgaaggttga accattggat tcactcagct tatttcatct
     1741 taaaacggaa tccaacggga aggcattcac tgataaagct tataattctc aggtacagtt
     1801 aacggtgaat gccaatcaga aagcccatcc tttgacccag ccctcctctc cacctaacca
     1861 gtgtgctaac gtgatggcag gcgatgacca aatacggttt cagcaggttg ttaaggagca
     1921 actcatgcat cagagactgc caacattgcc tggtatctct catgaaacac ccttaccgga
     1981 gtcagcacta actctcagga atgtaaatgt agtgtgttca ggtggaatta cagtggtttc
     2041 taccaaaagt gaagaggaag tctgttcatc cagttttgga acatcagaat tttccacagt
     2101 ggacagtgca cagaaaaatt ttaatgatta tgccatgaac ttctttacta accctacaaa
     2161 aaacctagtg tctataacta aagattctga actgcccacc tgcagctgtc ttgatcgagt
     2221 tatacaaaaa gacaaaggcc catattatac acaccttggg gcaggaccaa gtgttgctgc
     2281 tgtcagggaa atcatggaga ataggtatgg tcaaaaagga aacgcaataa ggatagaaat
     2341 agtagtgtac accggtaaag aagggaaaag ctctcatggg tgtccaattg ctaagtgggt
     2401 tttaagaaga agcagtgatg aagaaaaagt tctttgtttg gtccggcagc gtacaggcca
     2461 ccactgtcca actgctgtga tggtggtgct catcatggtg tgggatggca tccctcttcc
     2521 aatggccgac cggctataca cagagctcac agagaatcta aagtcataca atgggcaccc
     2581 taccgacaga agatgcaccc tcaatgaaaa tcgtacctgt acatgtcaag gaattgatcc
     2641 agagacttgt ggagcttcat tctcttttgg ctgttcatgg agtatgtact ttaatggctg
     2701 taagtttggt agaagcccaa gccccagaag atttagaatt gatccaagct ctcccttaca
     2761 tgaaaaaaac cttgaagata acttacagag tttggctaca cgattagctc caatttataa
     2821 gcagtatgct ccagtagctt accaaaatca ggtggaatat gaaaatgttg cccgagaatg
     2881 tcggcttggc agcaaggaag gtcgtccctt ctctggggtc actgcttgcc tggacttctg
     2941 tgcgcatccc tacagggcta ttcacaacat gaataatgga agcactgtgg tttgtacctt
     3001 aactcgagaa gataaccgct ctttgggtgt tattcctcaa gatgagcagc tccatgtgct
     3061 acctctttat aagctttcag acacagatga gtttggctcc aaggaaggaa tggaagccaa
     3121 gatcaaatct ggggccatcg aggtcctggc accccgccgc aaaaaaagaa cgtgtttcac
     3181 tcagcctgtt ccccgttctg gaaagaagag ggctgcgatg atgacagagg ttcttgcaca
     3241 taagataagg gcagtggaaa agaaacctat tccccgaatc aagcggaaga ataactcaac
     3301 aacaacaaac aacagtaagc cttcgtcact gccaacctta gggagtaaca ctgagaccgt
     3361 gcaacctgaa gtaaaaagtg aaaccgaacc ccattttatc ttaaaaagtt cagacaacac
     3421 taaaacttat tcgctgatgc catccgctcc tcacccagtg aaagaggcat ctccaggctt
     3481 ctcctggtcc ccgaagactg cttcagccac accagctcca ctgaagaatg acgcaacagc
     3541 ctcatgcggg ttttcagaaa gaagcagcac tccccactgt acgatgcctt cgggaagact
     3601 cagtggtgcc aatgcagctg ctgctgatgg ccctggcatt tcacagcttg gcgaagtggc
     3661 tcctctcccc accctgtctg ctcctgtgat ggagcccctc attaattctg agccttccac
     3721 tggtgtgact gagccgctaa cgcctcatca gccaaaccac cagccctcct tcctcacctc
     3781 tcctcaagac cttgcctctt ctccaatgga agaagatgag cagcattctg aagcagatga
     3841 gcctccatca gacgaacccc tatctgatga ccccctgtca cctgctgagg agaaattgcc
     3901 ccacattgat gagtattggt cagacagtga gcacatcttt ttggatgcaa atattggtgg
     3961 ggtggccatc gcacctgctc acggctcggt tttgattgag tgtgcccggc gagagctgca
     4021 cgctaccact cctgttgagc accccaaccg taatcatcca acccgcctct cccttgtctt
     4081 ttaccagcac aaaaacctaa ataagcccca acatggtttt gaactaaaca agattaagtt
     4141 tgaggctaaa gaagctaaga ataagaaaat gaaggcctca gagcaaaaag accaggcagc
     4201 taatgaaggt ccagaacagt cctctgaagt aaatgaattg aaccaaattc cttctcataa
     4261 agcattaaca ttaacccatg acaatgttgt caccgtgtcc ccttatgctc tcacacacgt
     4321 tgcggggccc tataaccatt gggtctgatc tagagggccc ttcgaaggta agcctatccc
     4381 taaccctctc ctcggtctcg attctacgcg taccggtcat catcaccatc accattgagt
     4441 ttaaacccgc tgatcagcct cgactgtgcc ttctagttgc cagccatctg ttgtttgccc
     4501 ctcccccgtg ccttccttga ccctggaagg tgccactccc actgtccttt cctaataaaa
     4561 tgaggaaatt gcatcgcatt gtctgagtag gtgtcattct attctggggg gtggggtggg
     4621 gcaggacagc aagggggagg attgggaaga caatagcagg catgctgggg atgcggtggg
     4681 ctctatggct tctgaggcgg aaagaaccag ctggggctct agggggtatc cccacgcgcc
     4741 ctgtagcggc gcattaagcg cggcgggtgt ggtggttacg cgcagcgtga ccgctacact
     4801 tgccagcgcc ctagcgcccg ctcctttcgc tttcttccct tcctttctcg ccacgttcgc
     4861 cggctttccc cgtcaagctc taaatcgggg gctcccttta gggttccgat ttagtgcttt
     4921 acggcacctc gaccccaaaa aacttgatta gggtgatggt tcacgtagtg ggccatcgcc
     4981 ctgatagacg gtttttcgcc ctttgacgtt ggagtccacg ttctttaata gtggactctt
     5041 gttccaaact ggaacaacac tcaaccctat ctcggtctat tcttttgatt tataagggat
     5101 tttgccgatt tcggcctatt ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa
     5161 ttaattctgt ggaatgtgtg tcagttaggg tgtggaaagt ccccaggctc cccagcaggc
     5221 agaagtatgc aaagcatgca tctcaattag tcagcaacca ggtgtggaaa gtccccaggc
     5281 tccccagcag gcagaagtat gcaaagcatg catctcaatt agtcagcaac catagtcccg
     5341 cccctaactc cgcccatccc gcccctaact ccgcccagtt ccgcccattc tccgccccat
     5401 ggctgactaa ttttttttat ttatgcagag gccgaggccg cctctgcctc tgagctattc
     5461 cagaagtagt gaggaggctt ttttggaggc ctaggctttt gcaaaaagct cccgggagct
     5521 tgtatatcca ttttcggatc tgatcaagag acaggatgag gatcgtttcg catgattgaa
     5581 caagatggat tgcacgcagg ttctccggcc gcttgggtgg agaggctatt cggctatgac
     5641 tgggcacaac agacaatcgg ctgctctgat gccgccgtgt tccggctgtc agcgcagggg
     5701 cgcccggttc tttttgtcaa gaccgacctg tccggtgccc tgaatgaact gcaggacgag
     5761 gcagcgcggc tatcgtggct ggccacgacg ggcgttcctt gcgcagctgt gctcgacgtt
     5821 gtcactgaag cgggaaggga ctggctgcta ttgggcgaag tgccggggca ggatctcctg
     5881 tcatctcacc ttgctcctgc cgagaaagta tccatcatgg ctgatgcaat gcggcggctg
     5941 catacgcttg atccggctac ctgcccattc gaccaccaag cgaaacatcg catcgagcga
     6001 gcacgtactc ggatggaagc cggtcttgtc gatcaggatg atctggacga agagcatcag
     6061 gggctcgcgc cagccgaact gttcgccagg ctcaaggcgc gcatgcccga cggcgaggat
     6121 ctcgtcgtga cccatggcga tgcctgcttg ccgaatatca tggtggaaaa tggccgcttt
     6181 tctggattca tcgactgtgg ccggctgggt gtggcggacc gctatcagga catagcgttg
     6241 gctacccgtg atattgctga agagcttggc ggcgaatggg ctgaccgctt cctcgtgctt
     6301 tacggtatcg ccgctcccga ttcgcagcgc atcgccttct atcgccttct tgacgagttc
     6361 ttctgagcgg gactctgggg ttcgcgaaat gaccgaccaa gcgacgccca acctgccatc
     6421 acgagatttc gattccaccg ccgccttcta tgaaaggttg ggcttcggaa tcgttttccg
     6481 ggacgccggc tggatgatcc tccagcgcgg ggatctcatg ctggagttct tcgcccaccc
     6541 caacttgttt attgcagctt ataatggtta caaataaagc aatagcatca caaatttcac
     6601 aaataaagca tttttttcac tgcattctag ttgtggtttg tccaaactca tcaatgtatc
     6661 ttataatgtc tgtataccgt cgacctctag ctagagcttg gcgtaatcat ggtcatagct
     6721 gtttcctgtg tgaaattgtt atccgctcac aattccacac aacatacgag ccggaagcat
     6781 aaagtgtaaa gcctggggtg cctaatgagt gagctaactc acattaattg cgttgcgctc
     6841 actgcccgct ttccagtcgg gaaacctgtc gtgccagctg cattaatgaa tcggccaacg
     6901 cgcggggaga ggcggtttgc gtattgggcg ctcttccgct tcctcgctca ctgactcgct
     6961 gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt
     7021 atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc aggaaccgta
     7081 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa
     7141 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc
     7201 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt
     7261 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca
     7321 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg
     7381 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat
     7441 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta
     7501 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct
     7561 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac
     7621 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa
     7681 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa
     7741 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt
     7801 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca
     7861 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca
     7921 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc
     7981 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa
     8041 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc
     8101 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca
     8161 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat
     8221 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag
     8281 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac
     8341 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt
     8401 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt
     8461 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc
     8521 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat
     8581 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca
     8641 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga
     8701 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg
     8761 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg
     8821 ttccgcgcac atttccccga aaagtgccac ctgacgt
//
