LOCUS       Exported                7751 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  Expresses the catalytic domain of TET1 in mammalian cells.
ACCESSION   .
VERSION     .
KEYWORDS    pIRES-hrGFP II-TET1_CD
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7751)
  AUTHORS   Jin C, Lu Y, Jelinek J, Liang S, Estecio MR, Barton MC, Issa JP
  TITLE     TET1 is a maintenance DNA demethylase that prevents methylation 
            spreading in differentiated cells.
  JOURNAL   Nucleic Acids Res. 2014 Jun;42(11):6956-71. doi: 10.1093/nar/gku372.
            Epub 2014 May 29.
  PUBMED    24875481
REFERENCE   2  (bases 1 to 7751)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7751
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     rep_origin      9..597
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     498..517
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     enhancer        742..1121
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        1122..1325
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     1370..1389
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1370..1388
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             3700..3771
                     /codon_start=1
                     /product="three tandem FLAG(R) epitope tags"
                     /label=3xFLAG
                     /translation="DYKDDDDKDYKDDDDKDYKDDDDK"
     misc_feature    3807..4379
                     /label=IRES
                     /note="internal ribosome entry site (IRES) of the 
                     encephalomyocarditis virus (EMCV)"
     primer_bind     complement(3974..3991)
                     /label=IRES reverse
                     /note="IRES internal ribosome entry site, reverse primer. 
                     Also called pCDH-rev"
     primer_bind     4201..4220
                     /label=IRES-F
                     /note="IRES internal ribosome entry site, forward primer"
     CDS             4385..5104
                     /codon_start=1
                     /product="humanized Renilla green fluorescent protein"
                     /label=hrGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKQILKNTGLQEIMSFKVNLEGVVNNHVFTMEGCGKGNILFGN
                     QLVQIRVTKGAPLPFAFDILSPAFQYGNRTFTKYPEDISDFFIQSFPAGFVYERTLRYE
                     DGGLVEIRSDINLIEGMFVYRVEYKGRNFPNDGPVMKKTITGLQPSFEVVYMNDGVLVG
                     QVILVYRLNSGKFYSCHMRTLMKSKGVVKDFPEYHFIQHRLEKTYVEDGGFVEQHETAI
                     AQLTSLGKPLGSLHEWV"
     primer_bind     complement(5135..5155)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(5135..5153)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(5173..5190)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    5179..5290
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     rep_origin      complement(5399..5854)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5536..5557)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     5748..5767
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     promoter        5881..5985
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(6010..6030)
                     /label=pBABE 3'
                     /note="SV40 enhancer, reverse primer for pBABE vectors"
     rep_origin      6175..6309
                     /label=SV40 ori
                     /note="SV40 origin of replication"
     primer_bind     6236..6255
                     /label=SV40pro-F
                     /note="SV40 promoter/origin, forward primer"
     CDS             6358..7152
                     /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
                     GLAPAELFARLKASMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIA
                     LATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     primer_bind     complement(6412..6431)
                     /label=Neo-R
                     /note="Neomycin resistance gene, reverse primer"
     primer_bind     7022..7041
                     /label=Neo-F
                     /note="Neomycin resistance gene, forward primer"
     primer_bind     complement(7340..7359)
                     /label=TK-pA-R
                     /note="Thymidine kinase polyA, reverse primer"
     polyA_signal    7384..7431
                     /label=HSV TK poly(A) signal
                     /note="herpes simplex virus thymidine kinase 
                     polyadenylation signal (Cole and Stacy, 1985)"
ORIGIN
        1 ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca
       61 ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta
      121 actggcttca gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc
      181 caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca
      241 gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta
      301 ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag
      361 cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt
      421 cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc
      481 acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac
      541 ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac
      601 gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc
      661 tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgccaggcg cgccgcgatg
      721 tacgggccag atttacgcgt tgacattgat tattgactag ttattaatag taatcaatta
      781 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      841 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      901 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      961 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
     1021 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
     1081 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
     1141 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
     1201 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
     1261 actccgcccc attgaagcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
     1321 gagctctctg gctaactaga gaacccactg cttactggct tatcgaaatt aatacgactc
     1381 actataggga gacccaagct tctggaggcc cgggctttca gggtaccgaa gaaggatcca
     1441 ctagtaacgg ccgccagtgt gctggaattc gcccttacca tggctaaaga ttctgaactg
     1501 cccacctgca gctgtcttga tcgagttata caaaaagaca aaggcccata ttatacacac
     1561 cttggggcag gaccaagtgt tgctgctgtc agggaaatca tggagaatag gtatggtcaa
     1621 aaaggaaacg caataaggat agaaatagta gtgtacaccg gtaaagaagg gaaaagctct
     1681 catgggtgtc caattgctaa gtgggtttta agaagaagca gtgatgaaga aaaagttctt
     1741 tgtttggtcc ggcagcgtac aggccaccac tgtccaactg ctgtgatggt ggtgctcatc
     1801 atggtgtggg atggcatccc tcttccaatg gccgaccggc tatacacaga gctcacagag
     1861 aatctaaagt catacaatgg gcaccctacc gacagaagat gcaccctcaa tgaaaatcgt
     1921 acctgtacat gtcaaggaat tgatccagag acttgtggag cttcattctc ttttggctgt
     1981 tcatggagta tgtactttaa tggctgtaag tttggtagaa gcccaagccc cagaagattt
     2041 agaattgatc caagctctcc cttacatgaa aaaaaccttg aagataactt acagagtttg
     2101 gctacacgat tagctccaat ttataagcag tatgctccag tagcttacca aaatcaggtg
     2161 gaatatgaaa atgttgcccg agaatgtcgg cttggcagca aggaaggtcg tcccttctct
     2221 ggggtcactg cttgcctgga cttctgtgct catccccaca gggacattca caacatgaat
     2281 aatggaagca ctgtggtttg taccttaact cgagaagata accgctcttt gggtgttatt
     2341 cctcaagatg agcagctcca tgtgctacct ctttataagc tttcagacac agatgagttt
     2401 ggctccaagg aaggaatgga agccaagatc aaatctgggg ccatcgaggt cctggcaccc
     2461 cgccgcaaaa aaagaacgtg tttcactcag cctgttcccc gttctggaaa gaagagggct
     2521 gcgatgatga cggaggttct tgcacataag ataagggcag tggaaaagaa acctattccc
     2581 cgaatcaagc ggaagaataa ctcaacaaca acaaacaaca gtaagccttc gtcactgcca
     2641 accttaggga gtaacactga gaccgtgcaa cctgaagtaa aaagtgaaac cgaaccccat
     2701 tttatcttaa aaagttcaga caacactaaa acttattcgc tgatgccatc cgctcctcac
     2761 ccagtgaaag aggcatctcc aggcttctcc tggtccccga agactgcttc agccacacca
     2821 gctccactga agaatgacgc aacagcctca tgcgggtttt cagaaagaag cagcactccc
     2881 cactgtacga tgccttcggg aagactcagt ggtgccaatg cagctgctgc tgatggccct
     2941 ggcatttcac agcttggcga agtggctcct ctccccaccc tgtctgctcc tgtgatggag
     3001 cccctcatta attctgagcc ttccactggt gtgactgagc cgctaacgcc tcatcagcca
     3061 aaccaccagc cctccttcct cacctctcct caagaccttg cctcttctcc aatggaagaa
     3121 gatgagcagc attctgaagc agatgagcct ccatcagacg aacccctatc tgatgacccc
     3181 ctgtcacctg ctgaggagaa attgccccac attgatgagt attggtcaga cagtgagcac
     3241 atctttttgg atgcaaatat tggtggggtg gccatcgcac ctgctcacgg ctcggttttg
     3301 attgagtgtg cccggcggga gctgcacgct accactcctg ttgagcaccc caaccgtaat
     3361 catccaaccc gcctctccct tgtcttttac cagcacaaaa acctaaataa gccccaacat
     3421 ggttttgaac taaacaagat taagtttgag gctaaagaag ctaagaataa gaaaatgaag
     3481 gcctcagagc aaaaagacca ggcagctaat gaaggtccag aacagtcctc tgaagtaaat
     3541 gaattgaacc aaattccttc tcataaagca ttaacattaa cccatgacaa tgttgtcacc
     3601 gtgtcccctt atgctctcac acacgttgcg gggccctata accattgggt ctgtaggata
     3661 agggcgaatt ctgcagatat ccatcacact ggcggccgcg actacaagga tgacgatgac
     3721 aaggattaca aagacgacga tgataaggac tataaggatg atgacgacaa ataatagcaa
     3781 ttcctcgacg actgcatagg gttacccccc tctccctccc ccccccctaa cgttactggc
     3841 cgaagccgct tggaataagg ccggtgtgcg tttgtctata tgttattttc caccatattg
     3901 ccgtcttttg gcaatgtgag ggcccggaaa cctggccctg tcttcttgac gagcattcct
     3961 aggggtcttt cccctctcgc caaaggaatg caaggtctgt tgaatgtcgt gaaggaagca
     4021 gttcctctgg aagcttcttg aagacaaaca acgtctgtag cgaccctttg caggcagcgg
     4081 aaccccccac ctggcgacag gtgcctctgc ggccaaaagc cacgtgtata agatacacct
     4141 gcaaaggcgg cacaacccca gtgccacgtt gtgagttgga tagttgtgga aagagtcaaa
     4201 tggctctcct caagcgtatt caacaagggg ctgaaggatg cccagaaggt accccattgt
     4261 atgggatctg atctggggcc tcggtgcaca tgctttacat gtgtttagtc gaggttaaaa
     4321 aacgtctagg ccccccgaac cacggggacg tggttttcct ttgaaaaaca cgatgataat
     4381 gaccatggtg agcaagcaga tcctgaagaa caccggcctg caggagatca tgagcttcaa
     4441 ggtgaacctg gagggcgtgg tgaacaacca cgtgttcacc atggagggct gcggcaaggg
     4501 caacatcctg ttcggcaacc agctggtgca gatccgcgtg accaagggcg cccccctgcc
     4561 cttcgccttc gacatcctga gccccgcctt ccagtacggc aaccgcacct tcaccaagta
     4621 ccccgaggac atcagcgact tcttcatcca gagcttcccc gccggcttcg tgtacgagcg
     4681 caccctgcgc tacgaggacg gcggcctggt ggagatccgc agcgacatca acctgatcga
     4741 ggggatgttc gtgtaccgcg tggagtacaa gggccgcaac ttccccaacg acggccccgt
     4801 gatgaagaag accatcaccg gcctgcagcc cagcttcgag gtggtgtaca tgaacgacgg
     4861 cgtgctggtg ggccaggtga tcctggtgta ccgcctgaac agcggcaagt tctacagctg
     4921 ccacatgcgc accctgatga agagcaaggg cgtggtgaag gacttccccg agtaccactt
     4981 catccagcac cgcctggaga agacctacgt ggaggacggc ggcttcgtag agcagcacga
     5041 gaccgccatc gcccagctga ccagcctggg caagcccctg ggcagcctgc acgagtgggt
     5101 gtaagctcga gcatgcatct agagggccct attcccttta gtgagggtta attgctagag
     5161 ctcgctgatc agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc
     5221 ccgtgccttc cttgaccctg gaaggtgcca ctctcactgt cctttcctaa taaaatgagg
     5281 aaattgcatc gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg
     5341 acagcaaggg ggaggattgg gaagacaata gcaggcatgc tggggcgccg gcgcgcgtaa
     5401 attgtaagcg ttaatatttt gttaaaattc gcgttaaatt tttgttaaat cagctcattt
     5461 tttaaccaat aggccgaaat cggcaaaatc ccttataaat caaaagaata gaccgagata
     5521 gggttgagtg ttgttccagt ttggaacaag agtccactat taaagaacgt ggactccaac
     5581 gtcaaagggc gaaaaaccgt ctatcagggc gatggcccac tacgtgaacc atcaccctaa
     5641 tcaagttttt tggggtcgag gtgccgtaaa gcactaaatc ggaaccctaa agggagcccc
     5701 cgatttagag cttgacgggg aaagccggcg aacgtggcga gaaaggaagg gaagaaagcg
     5761 aaaggagcgg gcgctagggc gctggcaagt gtagcggtca cgctgcgcgt aaccaccaca
     5821 cccgccgcgc ttaatgcgcc gctacagggc gcgtcaggtg gcacttttcg gggaaatgtg
     5881 cgcggaaccc ctatttgttt atttttctaa atacattcaa atatgtatcc gctcatgaga
     5941 caataaccct gataaatgct tcaataatat tgaaaaagga agaatcctga ggcggaaaga
     6001 accagctgtg gaatgtgtgt cagttagggt gtggaaagtc cccaggctcc ccagcaggca
     6061 gaagtatgca aagcatgcat ctcaattagt cagcaaccag gtgtggaaag tccccaggct
     6121 ccccagcagg cagaagtatg caaagcatgc atctcaatta gtcagcaacc atagtcccgc
     6181 ccctaactcc gcccagttcc gcccattctc cgccccatgg ctgactaatt ttttttattt
     6241 atgcagaggc cgaggccgcc tcggcctctg agctattcca gaagtagtga ggaggctttt
     6301 ttggaggcct aggcttttgc aaagatcgat caagagacag gatgaggatc gtttcgcatg
     6361 attgaacaag atggattgca cgcaggttct ccggccgctt gggtggagag gctattcggc
     6421 tatgactggg cacaacagac aatcggctgc tctgatgccg ccgtgttccg gctgtcagcg
     6481 caggggcgcc cggttctttt tgtcaagacc gacctgtccg gtgccctgaa tgaactgcaa
     6541 gacgaggcag cgcggctatc gtggctggcc acgacgggcg ttccttgcgc agctgtgctc
     6601 gacgttgtca ctgaagcggg aagggactgg ctgctattgg gcgaagtgcc ggggcaggat
     6661 ctcctgtcat ctcaccttgc tcctgccgag aaagtatcca tcatggctga tgcaatgcgg
     6721 cggctgcata cgcttgatcc ggctacctgc ccattcgacc accaagcgaa acatcgcatc
     6781 gagcgagcac gtactcggat ggaagccggt cttgtcgatc aggatgatct ggacgaagaa
     6841 catcaggggc tcgcgccagc cgaactgttc gccaggctca aggcgagcat gcccgacggc
     6901 gaggatctcg tcgtgaccca tggcgatgcc tgcttgccga atatcatggt ggaaaatggc
     6961 cgcttttctg gattcatcga ctgtggccgg ctgggtgtgg cggaccgcta tcaggacata
     7021 gcgttggcta cccgtgatat tgctgaagaa cttggcggcg aatgggctga ccgcttcctc
     7081 gtgctttacg gtatcgccgc tcccgattcg cagcgcatcg ccttctatcg ccttcttgac
     7141 gagttcttct gagcgggact ctggggttcg aaatgaccga ccaagcgacg cccaacctgc
     7201 catcacgaga tttcgattcc accgccgcct tctatgaaag gttgggcttc ggaatcgttt
     7261 tccgggacgc cggctggatg atcctccagc gcggggatct catgctggag ttcttcgccc
     7321 accctagggg gaggctaact gaaacacgga aggagacaat accggaagga acccgcgcta
     7381 tgacggcaat aaaaagacag aataaaacgc acggtgttgg gtcgtttgtt cataaacgcg
     7441 gggttcggtc ccagggctgg cactctgtcg ataccccacc gagaccccat tggggccaat
     7501 acgcccgcgt ttcttccttt tccccacccc accccccaag ttcgggtgaa ggcccagggc
     7561 tcgcagccaa cgtcggggcg gcaggccctg ccatagcctc aggttactca tatatacttt
     7621 agattgattt aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata
     7681 atctcatgac caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag
     7741 aaaagatcaa a
//
