LOCUS       Exported                6509 bp ds-DNA     circular SYN 27-APR-2023
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6509)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Friday, Apr 28, 2023 from SnapGene 5.0.8
            https://www.snapgene.com
COMMENT     pOTTC1751 was constructed by Chris Richie at NIDA/GEVVC (CR2177_D).
            Custom-linkered, V5-tagged, codon-optimized insert for estrogen 
            receptor ligand binding domain was produced by gBLOCK and cloned 
            into pcDNA3.1 (pOTTC735).
FEATURES             Location/Qualifiers
     source          1..6509
                     /organism="synthetic DNA construct"
                     /mol_type="other DNA"
     promoter        232..819
                     /label=CMV promoter (pcDNA3.1)
     enhancer        235..614
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        863..881
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     misc_recomb     901..916
                     /label=GEVVC NheI Infusion linker
     CDS             939..2009
                     /label=V5-ER-LBD
     misc_binding    939..2006
                     /label=V5-ERa
     misc_feature    981..1022
                     /label=V5 epitope [GKPIPNPLLGLDST]
     CDS             981..1022
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             1116..2009
                     /codon_start=1
                     /gene="ESR1"
                     /product="estrogen nuclear receptor alpha ligand-binding 
                     domain"
                     /label=ER-LBD
                     /translation="KRSKKNSLALSLTADQMVSALLDAEPPILYSEYDPTRPFSEASMM
                     GLLTNLADRELVHMINWAKRVPGFVDLTLHDQVHLLECAWLEILMIGLVWRSMEHPGKL
                     LFAPNLLLDRNQGKCVEGMVEIFDMLLATSSRFRMMNLQGEEFVCLKSIILLNSGVYTF
                     LSSTLKSLEEKDHIHRVLDKITDTLIHLMAKAGLTLQQQHQRLAQLLLILSHIRHMSNK
                     GMEHLYSMKCKNVVPLYDLLLEMLDAHRLHAPTSRGGASVEETDQSHLATAGSTSSHSL
                     QKYYITGEAEGFPATV"
     misc_recomb     2017..2033
                     /label=GEVVC AscI Infusion linker
     polyA_signal    2109..2333
                     /label=bGHpA
     rep_origin      2379..2807
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     rep_origin      2396..2702
                     /label=f1 origin
     variation       2427..2456
                     /label=SNP Found to end with T in CYB090_B
     variation       2682..2711
                     /label=SNP Found to end with T in CYB090_B
     variation       2731..2760
                     /label=SNP Found to end with T in CYB090_B
     promoter        2821..3150
                     /label=SV40 promoter
                     /note="SV40 enhancer and early promoter"
     promoter        2834..3142
                     /label=SV40 early promoter
     rep_origin      2849..3150
                     /label=SV40ori
     promoter        2954..3150
                     /label=SV40 early promoter
     CDS             3217..4011
                     /label=Neo(R)
                     /note="/invitrogen=1260000"
                     /note="/vntifkey=4"
     CDS             3220..4011
                     /codon_start=1
                     /label=KanR/NeoR
                     /translation="IEQDGLHAGSPAAWVERLFGYDWAQQTIGCSDAAVFRLSAQGRPV
                     LFVKTDLSGALNELQDEAARLSWLATTGVPCAAVLDVVTEAGRDWLLLGEVPGQDLLSS
                     HLAPAEKVSIMADAMRRLHTLDPATCPFDHQAKHRIERARTRMEAGLVDQDDLDEEHQG
                     LAPAELFARLKARMPDGEDLVVTHGDACLPNIMVENGRFSGFIDCGRLGVADRYQDIAL
                     ATRDIAEELGGEWADRFLVLYGIAAPDSQRIAFYRLLDEFF"
     polyA_signal    4185..4315
                     /label=SV40 pA
                     /label=SV40\pA
                     /note="/invitrogen=1840000"
                     /note="/vntifkey=25"
     polyA_signal    4185..4306
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4351..4371)
                     /label=M13-rev
     misc_binding    complement(4377..4399)
                     /label=LacO
     protein_bind    4379..4395
                     /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(4403..4433)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4448..4469
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     rep_origin      complement(4698..5368)
                     /direction=LEFT
                     /label=pUC origin
     rep_origin      4739..5418
                     /locus_tag="ColE1 origin"
                     /label=ColE1 origin
                     /note="Plasmid origin of replication"
     rep_origin      4742..5358
                     /label=pBR322 origin
     CDS             complement(5516..6373)
                     /label=AmpR
     promoter        complement(6374..6478)
                     /gene="bla"
                     /label=AmpR promoter
     promoter        complement(6374..6472)
                     /label=Bla promoter (prokaryotic)
ORIGIN
        1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg
       61 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg
      121 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc
      181 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt
      241 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata
      301 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc
      361 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc
      421 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt
      481 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt
      541 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca
      601 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg
      661 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc
      721 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg
      781 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca
      841 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctaga
      901 cgaagttatc gctagcgcta ccggactcag atctcaccat ggaagcttac tgtcatatta
      961 aggggttcga tgccgtgaat ggaaagccaa tacccaatcc tctgctgggc ttggactcaa
     1021 ctagaaccgg cggcggttgt ccatgggcca ctggttcagg ttcatctgct ggagacatga
     1081 gagctgccaa cctttggcca agcccgctca tgatcaaacg ctctaagaag aacagcctgg
     1141 ccttgtccct gacggccgac cagatggtca gtgccttgtt ggatgctgag ccccccatac
     1201 tctattccga gtatgatcct accagaccct tcagtgaagc ctccatgatg ggcttactga
     1261 ccaacctggc agacagggag ctggttcaca tgatcaactg ggcgaagagg gtgccaggct
     1321 ttgtggattt gaccctccat gatcaggtcc accttctgga atgtgcctgg ctagagatcc
     1381 tgatgattgg cctcgtctgg cgctccatgg agcacccagg gaagctactg tttgctccta
     1441 acttgctctt ggacaggaac cagggaaaat gtgtagaggg catggtggag atcttcgaca
     1501 tgctgctggc tacatcatct cggttccgca tgatgaatct gcagggagag gagtttgtgt
     1561 gcctcaaatc tattattttg cttaattctg gagtgtacac atttctgtcc agcaccctga
     1621 agtctctgga agagaaggac catatccacc gagtcctgga caagatcaca gacactttga
     1681 tccacctgat ggccaaggca ggcctgaccc tgcagcagca gcaccagcgg ctggcccagc
     1741 tcctcctcat cctctcccac atcaggcaca tgagtaacaa aggcatggag catctgtaca
     1801 gcatgaagtg caagaacgtg gtgcccctct atgacctgct gctggagatg ctggacgccc
     1861 accgcctaca tgcgcccact agccgtggag gggcatccgt ggaggagacg gaccaaagcc
     1921 acttggccac tgcgggctct acttcatcgc attccttgca aaagtattac atcacggggg
     1981 aggcagaggg tttccctgcc acagtctagg caggtgggcg cgccataact tcgaattctg
     2041 cagatatcca gcacagtggc ggccgctcga gtctagaggg cccgtttaaa cccgctgatc
     2101 agcctcgact gtgccttcta gttgccagcc atctgttgtt tgcccctccc ccgtgccttc
     2161 cttgaccctg gaaggtgcca ctcccactgt cctttcctaa taaaatgagg aaattgcatc
     2221 gcattgtctg agtaggtgtc attctattct ggggggtggg gtggggcagg acagcaaggg
     2281 ggaggattgg gaagacaata gcaggcatgc tggggatgcg gtgggctcta tggcttctga
     2341 ggcggaaaga accagctggg gctctagggg gtatccccac gcgccctgta gcggcgcatt
     2401 aagcgcggcg ggtgtggtgg ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
     2461 gcccgctcct ttcgctttct tcccttcctt tctcgccacg ttcgccggct ttccccgtca
     2521 agctctaaat cgggggctcc ctttagggtt ccgatttagt gctttacggc acctcgaccc
     2581 caaaaaactt gattagggtg atggttcacg tagtgggcca tcgccctgat agacggtttt
     2641 tcgccctttg acgttggagt ccacgttctt taatagtgga ctcttgttcc aaactggaac
     2701 aacactcaac cctatctcgg tctattcttt tgatttataa gggattttgc cgatttcggc
     2761 ctattggtta aaaaatgagc tgatttaaca aaaatttaac gcgaattaat tctgtggaat
     2821 gtgtgtcagt tagggtgtgg aaagtcccca ggctccccag caggcagaag tatgcaaagc
     2881 atgcatctca attagtcagc aaccaggtgt ggaaagtccc caggctcccc agcaggcaga
     2941 agtatgcaaa gcatgcatct caattagtca gcaaccatag tcccgcccct aactccgccc
     3001 atcccgcccc taactccgcc cagttccgcc cattctccgc cccatggctg actaattttt
     3061 tttatttatg cagaggccga ggccgcctct gcctctgagc tattccagaa gtagtgagga
     3121 ggcttttttg gaggcctagg cttttgcaaa aagctcccgg gagcttgtat atccattttc
     3181 ggatctgatc aagagacagg atgaggatcg tttcgcatga ttgaacaaga tggattgcac
     3241 gcaggttctc cggccgcttg ggtggagagg ctattcggct atgactgggc acaacagaca
     3301 atcggctgct ctgatgccgc cgtgttccgg ctgtcagcgc aggggcgccc ggttcttttt
     3361 gtcaagaccg acctgtccgg tgccctgaat gaactgcagg acgaggcagc gcggctatcg
     3421 tggctggcca cgacgggcgt tccttgcgca gctgtgctcg acgttgtcac tgaagcggga
     3481 agggactggc tgctattggg cgaagtgccg gggcaggatc tcctgtcatc tcaccttgct
     3541 cctgccgaga aagtatccat catggctgat gcaatgcggc ggctgcatac gcttgatccg
     3601 gctacctgcc cattcgacca ccaagcgaaa catcgcatcg agcgagcacg tactcggatg
     3661 gaagccggtc ttgtcgatca ggatgatctg gacgaagagc atcaggggct cgcgccagcc
     3721 gaactgttcg ccaggctcaa ggcgcgcatg cccgacggcg aggatctcgt cgtgacccat
     3781 ggcgatgcct gcttgccgaa tatcatggtg gaaaatggcc gcttttctgg attcatcgac
     3841 tgtggccggc tgggtgtggc ggaccgctat caggacatag cgttggctac ccgtgatatt
     3901 gctgaagagc ttggcggcga atgggctgac cgcttcctcg tgctttacgg tatcgccgct
     3961 cccgattcgc agcgcatcgc cttctatcgc cttcttgacg agttcttctg agcgggactc
     4021 tggggttcga aatgaccgac caagcgacgc ccaacctgcc atcacgagat ttcgattcca
     4081 ccgccgcctt ctatgaaagg ttgggcttcg gaatcgtttt ccgggacgcc ggctggatga
     4141 tcctccagcg cggggatctc atgctggagt tcttcgccca ccccaacttg tttattgcag
     4201 cttataatgg ttacaaataa agcaatagca tcacaaattt cacaaataaa gcattttttt
     4261 cactgcattc tagttgtggt ttgtccaaac tcatcaatgt atcttatcat gtctgtatac
     4321 cgtcgacctc tagctagagc ttggcgtaat catggtcata gctgtttcct gtgtgaaatt
     4381 gttatccgct cacaattcca cacaacatac gagccggaag cataaagtgt aaagcctggg
     4441 gtgcctaatg agtgagctaa ctcacattaa ttgcgttgcg ctcactgccc gctttccagt
     4501 cgggaaacct gtcgtgccag ctgcattaat gaatcggcca acgcgcgggg agaggcggtt
     4561 tgcgtattgg gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
     4621 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca gaatcagggg
     4681 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg
     4741 ccgcgttgct ggcgtttttc cataggctcc gcccccctga cgagcatcac aaaaatcgac
     4801 gctcaagtca gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg
     4861 gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
     4921 ttctcccttc gggaagcgtg gcgctttctc atagctcacg ctgtaggtat ctcagttcgg
     4981 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct
     5041 gcgccttatc cggtaactat cgtcttgagt ccaacccggt aagacacgac ttatcgccac
     5101 tggcagcagc cactggtaac aggattagca gagcgaggta tgtaggcggt gctacagagt
     5161 tcttgaagtg gtggcctaac tacggctaca ctagaagaac agtatttggt atctgcgctc
     5221 tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
     5281 ccgctggtag cggttttttt gtttgcaagc agcagattac gcgcagaaaa aaaggatctc
     5341 aagaagatcc tttgatcttt tctacggggt ctgacgctca gtggaacgaa aactcacgtt
     5401 aagggatttt ggtcatgaga ttatcaaaaa ggatcttcac ctagatcctt ttaaattaaa
     5461 aatgaagttt taaatcaatc taaagtatat atgagtaaac ttggtctgac agttaccaat
     5521 gcttaatcag tgaggcacct atctcagcga tctgtctatt tcgttcatcc atagttgcct
     5581 gactccccgt cgtgtagata actacgatac gggagggctt accatctggc cccagtgctg
     5641 caatgatacc gcgagaccca cgctcaccgg ctccagattt atcagcaata aaccagccag
     5701 ccggaagggc cgagcgcaga agtggtcctg caactttatc cgcctccatc cagtctatta
     5761 attgttgccg ggaagctaga gtaagtagtt cgccagttaa tagtttgcgc aacgttgttg
     5821 ccattgctac aggcatcgtg gtgtcacgct cgtcgtttgg tatggcttca ttcagctccg
     5881 gttcccaacg atcaaggcga gttacatgat cccccatgtt gtgcaaaaaa gcggttagct
     5941 ccttcggtcc tccgatcgtt gtcagaagta agttggccgc agtgttatca ctcatggtta
     6001 tggcagcact gcataattct cttactgtca tgccatccgt aagatgcttt tctgtgactg
     6061 gtgagtactc aaccaagtca ttctgagaat agtgtatgcg gcgaccgagt tgctcttgcc
     6121 cggcgtcaat acgggataat accgcgccac atagcagaac tttaaaagtg ctcatcattg
     6181 gaaaacgttc ttcggggcga aaactctcaa ggatcttacc gctgttgaga tccagttcga
     6241 tgtaacccac tcgtgcaccc aactgatctt cagcatcttt tactttcacc agcgtttctg
     6301 ggtgagcaaa aacaggaagg caaaatgccg caaaaaaggg aataagggcg acacggaaat
     6361 gttgaatact catactcttc ctttttcaat attattgaag catttatcag ggttattgtc
     6421 tcatgagcgg atacatattt gaatgtattt agaaaaataa acaaataggg gttccgcgca
     6481 catttccccg aaaagtgcca cctgacgtc
//
