LOCUS       Exported                7766 bp ds-DNA     circular SYN 12-MAY-2021
DEFINITION  bacterial expression of human LIG3.
ACCESSION   .
VERSION     .
KEYWORDS    pGEX4T-hLIG3
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7766)
  AUTHORS   Weber AR, Krawczyk C, Robertson AB, Kusnierczyk A, Vagbo CB, 
            Schuermann D, Klungland A, Schar P
  TITLE     Biochemical reconstitution of TET1-TDG-BER-dependent active DNA 
            demethylation reveals a highly coordinated mechanism.
  JOURNAL   Nat Commun. 2016 Mar 2;7:10806. doi: 10.1038/ncomms10806.
  PUBMED    26932196
REFERENCE   2  (bases 1 to 7766)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 12, 2021 from SnapGene Server 1.1.58
            http://www.snapgene.com
FEATURES             Location/Qualifiers
     source          1..7766
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        93..121
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    129..145
                     /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)."
     CDS             168..821
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     primer_bind     779..801
                     /label=pGEX 5'
                     /note="pGEX vectors, Glutathione-S-transferase, forward 
                     primer"
     CDS             828..845
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             864..890
                     /codon_start=1
                     /product="HA (human influenza hemagglutinin) epitope tag"
                     /label=HA
                     /translation="YPYDVPDYA"
     regulatory      1444..1453
                     /regulatory_class="other"
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
     primer_bind     complement(3726..3748)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3893..3911)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        3979..4083
                     /gene="bla"
                     /label=AmpR promoter
     CDS             4084..4944
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(4302..4321)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5115..5703
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5604..5623
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5857..5874
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        5947..6024
                     /gene="lacI (mutant)"
                     /label=lacIq promoter
                     /note="In the lacIq allele, a single base change in the 
                     promoter boosts expression of the lacI gene about 10-fold."
     CDS             6025..7107
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=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)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(6044..6063)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     protein_bind    7120..7141
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        7156..7186
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    7194..7210
                     /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)."
     primer_bind     7218..7234
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     7218..7234
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             7230..7757
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGITLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(7250..7267)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(7250..7266)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(7259..7281)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 taattcgtgt cgctcaaggc gcactcccgt tctggataat gttttttgcg ccgacatcat
       61 aacggttctg gcaaatattc tgaaatgagc tgttgacaat taatcatcgg ctcgtataat
      121 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agtattcatg tcccctatac
      181 taggttattg gaaaattaag ggccttgtgc aacccactcg acttcttttg gaatatcttg
      241 aagaaaaata tgaagagcat ttgtatgagc gcgatgaagg tgataaatgg cgaaacaaaa
      301 agtttgaatt gggtttggag tttcccaatc ttccttatta tattgatggt gatgttaaat
      361 taacacagtc tatggccatc atacgttata tagctgacaa gcacaacatg ttgggtggtt
      421 gtccaaaaga gcgtgcagag atttcaatgc ttgaaggagc ggttttggat attagatacg
      481 gtgtttcgag aattgcatat agtaaagact ttgaaactct caaagttgat tttcttagca
      541 agctacctga aatgctgaaa atgttcgaag atcgtttatg tcataaaaca tatttaaatg
      601 gtgatcatgt aacccatcct gacttcatgt tgtatgacgc tcttgatgtt gttttataca
      661 tggacccaat gtgcctggat gcgttcccaa aattagtttg ttttaaaaaa cgtattgaag
      721 ctatcccaca aattgataag tacttgaaat ccagcaagta tatagcatgg cctttgcagg
      781 gctggcaagc cacgtttggt ggtggcgacc atcctccaaa atcggatctg gttccgcgtg
      841 gatccccgaa ttcccgggtc gactacccat acgatgttcc agattacgct cttatggctg
      901 agcaacggtt ctgtgtggac tatgccaagc gtggcacagc tggctgcaaa aaatgcaagg
      961 aaaagattgt gaagggcgta tgccgaattg gcaaagtggt gcccaatccc ttctcagagt
     1021 ctgggggtga tatgaaagag tggtaccaca ttaaatgcat gtttgagaaa ctagagcggg
     1081 cccgggccac cacaaaaaaa atcgaggacc tcacagagct ggaaggctgg gaagagctgg
     1141 aagataatga gaaggaacag ataacccagc acattgcaga tctgtcttct aaggcagcag
     1201 gtacaccaaa gaagaaagct gttgtccagg ctaagttgac aaccactggc caggtgactt
     1261 ctccagtgaa aggcgcctca tttgtcacca gtaccaatcc ccggaaattt tctggctttt
     1321 cagccaagcc caacaactct ggggaagccc cctcgagccc cacccctaag agaagtctgt
     1381 cttcaagcaa atgtgacccc aggcataagg actgtctgct acgggagttt cgaaagttat
     1441 gcgccatggt ggccgataat cctagctaca acacgaagac ccagatcatc caggacttcc
     1501 ttcggaaagg ctcagcagga gatggtttcc acggtgatgt gtacctaaca gtgaagctgc
     1561 tgctgccagg agtcattaag actgtttaca acttgaacga taagcagatt gtgaagcttt
     1621 tcagtcgcat ttttaactgc aacccagatg atatggcacg ggacctagag cagggtgacg
     1681 tgtcagagac aatcagagtc ttctttgagc agagcaagtc tttcccccca gctgccaaga
     1741 gcctccttac catccaggaa gtggatgagt tccttctgcg gctgtccaag ctcaccaagg
     1801 aggatgagca gcaacaggcc ctacaggaca ttgcctccag gtgtacagcc aatgacctta
     1861 aatgcatcat caggttgatc aaacatgatc tgaagatgaa ctcaggtgca aaacatgtgt
     1921 tagacgccct tgaccccaat gcctatgaag ccttcaaagc ctcgcgcaac ctgcaggatg
     1981 tggtggagcg ggtccttcac aacgcgcagg aggtggagaa tgagccgggc cagagacgag
     2041 ctctgagcgt ccaggcctcg ctgatgacac ctgtgcagcc catgttggcg gaggcctgca
     2101 agtccgttga gtatgcaatg aagaaatgtc ccaatggcat gttctctgag atcaagtacg
     2161 atggagagcg agtccaggtg cataagaatg gagaccactt cagctacttc agccgcagtc
     2221 tcaagcccgt ccttcctcac aaggtggccc actttaagga ctacattccc caggcttttc
     2281 ctgggggcca cagcatgatc ttggattctg aagtgcttct gattgacaac aagacaggca
     2341 aaccactgcc ctttgggact ctgggagtac acaagaaagc agccttccag gatgctaatg
     2401 tctgcctgtt tgtttttgat tgtatctact ttaatgatgt cagcttgatg gacagacctc
     2461 tgtgtgagcg gcggaagttt cttcatgaca acatggttga aattccaaac cggatcatgt
     2521 tctcagaaat gaagcgagtc acaaaagctt tggacttggc tgacatgata acccgggtga
     2581 tccaggaggg attggagggg ctggtgctga aggatgtgaa gggtacatat gagcctggga
     2641 agcggcactg gctgaaagtg aagaaagact atttgaacga gggggccatg gccgacacag
     2701 ctgacctggt ggtccttgga gccttctatg ggcaagggag caaaggcggc atgatgtcaa
     2761 tcttcctcat gggctgctac gaccctggca gccagaagtg gtgcacagtc accaagtgtg
     2821 caggaggcca tgatgatgcc acgcttgccc gcctgcagaa tgaactagac atggtgaaga
     2881 tcagcaagga ccccagcaaa atacccagct ggttgaaggt caacaagatc tactatcctg
     2941 acttcatcgt cccagaccca aagaaagctg ccgtgtggga gatcacaggg gctgaattct
     3001 ccaaatcgga ggctcataca gctgacggga tctccatccg attccctcgc tgcacccgaa
     3061 tccgagatga taaggactgg aaatctgcca ctaaccttcc ccaactcaag gaactgtacc
     3121 agttgtccaa ggagaaggca gacttcactg tagtggctgg agatgagggg agctccacta
     3181 cagggggtag cagtgaagag aataagggtc cctcagggtc tgctgtgtcc cgcaaggccc
     3241 ccagcaagcc ctcagccagt accaagaaag cagaagggaa gctgagtaac tccaacagca
     3301 aagatggcaa catgcagact gcaaagcctt ccgctatgaa ggtgggggag aagctggcca
     3361 caaagtcttc tccagtgaaa gtaggggaga agcggaaagc tgctgatgag acgctgtgcc
     3421 aaacaaaggt attgctggac atcttcactg gggtgcggct ttacttgcca ccctccacac
     3481 cagacttcag ccgtctcaga cgctactttg tggcattcga cggggacctg gtacaggaat
     3541 ttgatatgac ttcagccacg cacgtgctgg gtagcaggga caagaaccct gcggcccagc
     3601 aggtctcccc agagtggatt tgggcatgta tccggaaacg gagactggta gctccctgct
     3661 agtaagcggc cgcatcgtga ctgactgacg atctgcctcg cgcgtttcgg tgatgacggt
     3721 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc
     3781 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggcgcagcc
     3841 atgacccagt cacgtagcga tagcggagtg tataattctt gaagacgaaa gggcctcgtg
     3901 atacgcctat ttttataggt taatgtcatg ataataatgg tttcttagac gtcaggtggc
     3961 acttttcggg gaaatgtgcg cggaacccct atttgtttat ttttctaaat acattcaaat
     4021 atgtatccgc tcatgagaca ataaccctga taaatgcttc aataatattg aaaaaggaag
     4081 agtatgagta ttcaacattt ccgtgtcgcc cttattccct tttttgcggc attttgcctt
     4141 cctgtttttg ctcacccaga aacgctggtg aaagtaaaag atgctgaaga tcagttgggt
     4201 gcacgagtgg gttacatcga actggatctc aacagcggta agatccttga gagttttcgc
     4261 cccgaagaac gttttccaat gatgagcact tttaaagttc tgctatgtgg cgcggtatta
     4321 tcccgtgttg acgccgggca agagcaactc ggtcgccgca tacactattc tcagaatgac
     4381 ttggttgagt actcaccagt cacagaaaag catcttacgg atggcatgac agtaagagaa
     4441 ttatgcagtg ctgccataac catgagtgat aacactgcgg ccaacttact tctgacaacg
     4501 atcggaggac cgaaggagct aaccgctttt ttgcacaaca tgggggatca tgtaactcgc
     4561 cttgatcgtt gggaaccgga gctgaatgaa gccataccaa acgacgagcg tgacaccacg
     4621 atgcctgcag caatggcaac aacgttgcgc aaactattaa ctggcgaact acttactcta
     4681 gcttcccggc aacaattaat agactggatg gaggcggata aagttgcagg accacttctg
     4741 cgctcggccc ttccggctgg ctggtttatt gctgataaat ctggagccgg tgagcgtggg
     4801 tctcgcggta tcattgcagc actggggcca gatggtaagc cctcccgtat cgtagttatc
     4861 tacacgacgg ggagtcaggc aactatggat gaacgaaata gacagatcgc tgagataggt
     4921 gcctcactga ttaagcattg gtaactgtca gaccaagttt actcatatat actttagatt
     4981 gatttaaaac ttcattttta atttaaaagg atctaggtga agatcctttt tgataatctc
     5041 atgaccaaaa tcccttaacg tgagttttcg ttccactgag cgtcagaccc cgtagaaaag
     5101 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     5161 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     5221 aaggtaactg gcttcagcag agcgcagata ccaaatactg tccttctagt gtagccgtag
     5281 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     5341 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     5401 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     5461 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     5521 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     5581 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     5641 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     5701 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     5761 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     5821 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     5881 gaagagcgcc tgatgcggta ttttctcctt acgcatctgt gcggtatttc acaccgcata
     5941 aattccgaca ccatcgaatg gtgcaaaacc tttcgcggta tggcatgata gcgcccggaa
     6001 gagagtcaat tcagggtggt gaatgtgaaa ccagtaacgt tatacgatgt cgcagagtat
     6061 gccggtgtct cttatcagac cgtttcccgc gtggtgaacc aggccagcca cgtttctgcg
     6121 aaaacgcggg aaaaagtgga agcggcgatg gcggagctga attacattcc caaccgcgtg
     6181 gcacaacaac tggcgggcaa acagtcgttg ctgattggcg ttgccacctc cagtctggcc
     6241 ctgcacgcgc cgtcgcaaat tgtcgcggcg attaaatctc gcgccgatca actgggtgcc
     6301 agcgtggtgg tgtcgatggt agaacgaagc ggcgtcgaag cctgtaaagc ggcggtgcac
     6361 aatcttctcg cgcaacgcgt cagtgggctg atcattaact atccgctgga tgaccaggat
     6421 gccattgctg tggaagctgc ctgcactaat gttccggcgt tatttcttga tgtctctgac
     6481 cagacaccca tcaacagtat tattttctcc catgaagacg gtacgcgact gggcgtggag
     6541 catctggtcg cattgggtca ccagcaaatc gcgctgttag cgggcccatt aagttctgtc
     6601 tcggcgcgtc tgcgtctggc tggctggcat aaatatctca ctcgcaatca aattcagccg
     6661 atagcggaac gggaaggcga ctggagtgcc atgtccggtt ttcaacaaac catgcaaatg
     6721 ctgaatgagg gcatcgttcc cactgcgatg ctggttgcca acgatcagat ggcgctgggc
     6781 gcaatgcgcg ccattaccga gtccgggctg cgcgttggtg cggatatctc ggtagtggga
     6841 tacgacgata ccgaagacag ctcatgttat atcccgccgt taaccaccat caaacaggat
     6901 tttcgcctgc tggggcaaac cagcgtggac cgcttgctgc aactctctca gggccaggcg
     6961 gtgaagggca atcagctgtt gcccgtctca ctggtgaaaa gaaaaaccac cctggcgccc
     7021 aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct ggcacgacag
     7081 gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt agctcactca
     7141 ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg gaattgtgag
     7201 cggataacaa tttcacacag gaaacagcta tgaccatgat tacggattca ctggccgtcg
     7261 ttttacaacg tcgtgactgg gaaaaccctg gcgttaccca acttaatcgc cttgcagcac
     7321 atcccccttt cgccagctgg cgtaatagcg aagaggcccg caccgatcgc ccttcccaac
     7381 agttgcgcag cctgaatggc gaatggcgct ttgcctggtt tccggcacca gaagcggtgc
     7441 cggaaagctg gctggagtgc gatcttcctg aggccgatac tgtcgtcgtc ccctcaaact
     7501 ggcagatgca cggttacgat gcgcccatct acaccaacgt aacctatccc attacggtca
     7561 atccgccgtt tgttcccacg gagaatccga cgggttgtta ctcgctcaca tttaatgttg
     7621 atgaaagctg gctacaggaa ggccagacgc gaattatttt tgatggcgtt ggaattacgt
     7681 tatcgactgc acggtgcacc aatgcttctg gcgtcaggca gccatcggaa gctgtggtat
     7741 ggctgtgcag gtcgtaaatc actgca
//
