LOCUS       sequence-12935-e        7238 bp    DNA     circular SYN 15-APR-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7238)
  AUTHORS   Zhao Y, Terry D, Shi L, Weinstein H, Blanchard SC, Javitch JA
  TITLE     Single-molecule dynamics of gating in a neurotransmitter transporter
            homologue.
  JOURNAL   Nature. 2010 May 13. 465(7295):188-93.
  PUBMED    20463731
REFERENCE   2  (bases 1 to 7238)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Apr 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "Nature. 
            2010 May 13. 465(7295):188-93."
COMMENT     Sequence Label: pETO18G-H7C/R86C
FEATURES             Location/Qualifiers
     source          1..7238
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     20..39
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        20..38
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     protein_bind    39..63
                     /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)."
     RBS             78..100
                     /note="efficient ribosome binding site from bacteriophage 
                     T7 gene 10 (Olins and Rangwala, 1989)"
     CDS             1652..1669
                     /codon_start=1
                     /product="thrombin recognition and cleavage site"
                     /label=thrombin site
                     /translation="LVPRGS"
     CDS             1673..1696
                     /codon_start=1
                     /product="8xHis affinity tag"
                     /label=8xHis
                     /translation="HHHHHHHH"
     primer_bind     complement(1755..1773)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1769..1816
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(1991..1996,1997..2013,2014..2019))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2046..2064)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2132..2236
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2237..2305,2306..3097)
                     /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(2455..2474)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3268..3856
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3757..3776
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4010..4027
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4042..4184
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4110..4129)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4270..4292
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4286..4477)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5789..6871)
                     /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     6833..6852
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6872..6949)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7155..7174
                     /label=pBRrevBam
                     /note="pBR322 vectors, tet region, downstream of BamHI, 
                     reverse primer"
ORIGIN
        1 gatctcgatc ccgcgaaatt aatacgactc actatagggg aattgtgagc ggataacaat
       61 tcccctctag aaataatttt gtttaacttt aagaaggaga tataccatgg aagtgaaacg
      121 cgaatgttgg gcgacccgcc tgggcctgat tctggcgatg gcgggcaacg cggtgggcct
      181 gggcaacttt ctgcgctttc ctgtacaggc ggcggaaaac ggcggcggcg cgtttatgat
      241 tccgtatatt attgcgtttc tgctggtggg cattccgctg atgtggattg aatgggcgat
      301 gggccgctat ggcggcgcgc agggccacgg caccacacct gcgatttttt atctgctgtg
      361 gtgcaaccgc tttgcgaaaa ttctgggcgt gtttggcctg tggattccgc tggtggtggc
      421 gatttattat gtgtatattg aaagctggac cctgggcttt gcgattaaat ttctggtggg
      481 cctggtgccg gaaccgccgc cgaacgcgac ggatccggat agcattctgc gcccgtttaa
      541 agaatttctg tatagctata ttggcgtgcc gaaaggcgat gaaccgattc tgaaaccgag
      601 cctgtttgcg tatattgtgt ttctgattac catgtttatt aacgtgagca ttctgattcg
      661 cggcattagc aaaggcattg agcgctttgc gaaaattgcg atgccgaccc tgtttattct
      721 ggcggtgttt ctggtgattc gcgtgtttct gctggaaacc ccgaacggca ccgctgcaga
      781 tggcctgaac tttctgtgga ccccggattt tgaaaaactg aaagatccgg gcgtgtggat
      841 tgcggcggtg ggccagattt tttttaccct gagcctgggc tttggcgcga ttattaccta
      901 tgcgagctat gtacgtaaag atcaggatat tgtgctgagc ggcctgaccg cggcgaccct
      961 gaacgaaaaa gcggaagtga ttctgggcgg cagcattagc attccggcgg cggtggcgtt
     1021 ttttggcgtg gcgaacgcgg tggcgattgc gaaagcgggc gcgtttaacc taggctttat
     1081 taccctgccg gcgattttta gccagaccgc gggcggcacc tttctgggct ttctgtggtt
     1141 ttttctgctg ttttttgcgg gcctgaccag cagcattgcg attatgcagc cgatgattgc
     1201 gtttctggaa gatgaactta agctgagccg caaacatgcg gtgctgtgga ccgcggcgat
     1261 tgtgtttttt agcgcgcatc tggtgatgtt tctgaacaaa agcctggatg aaatggattt
     1321 ttgggcgggt accattggcg tggtgttttt tggcctgacc gaactgatta tttttttttg
     1381 gatttttggc gcggataaag cgtgggagga gattaaccgc ggcggcatta ttaaagtgcc
     1441 gcgcatttat tattatgtga tgcgctatat taccccggcg tttctggcgg tgctgctggt
     1501 ggtgtgggcc cgggaatata ttccgaaaat tatggaagaa acccattgga ccgtgtggat
     1561 tacccgcttt tatattattg gcctgtttct gtttctgacc tttctggtgt ttctggcgga
     1621 acgccgccgc aaccatgaaa gcgcgggcac cctggtgccg cgcggcagcg gtcatcatca
     1681 ccatcatcac catcattgac tcgaggatcc ggctgctaac aaagcccgaa aggaagctga
     1741 gttggctgct gccaccgctg agcaataact agcataaccc cttggggcct ctaaacgggt
     1801 cttgaggggt tttttgctga aaggaggaac tatatccgga tatcccgcaa gaggcccggc
     1861 agtaccggca taaccaagcc tatgcctaca gcatccaggg tgacggtgcc gaggatgacg
     1921 atgagcgcat tgttagattt catacacggt gcctgactgc gttagcaatt taactgtgat
     1981 aaactaccgc attaaagctt atcgatgata agctgtcaaa catgagaatt cttgaagacg
     2041 aaagggcctc gtgatacgcc tatttttata ggttaatgtc atgataataa tggtttctta
     2101 gacgtcaggt ggcacttttc ggggaaatgt gcgcggaacc cctatttgtt tatttttcta
     2161 aatacattca aatatgtatc cgctcatgag acaataaccc tgataaatgc ttcaataata
     2221 ttgaaaaagg aagagtatga gtattcaaca tttccgtgtc gcccttattc ccttttttgc
     2281 ggcattttgc cttcctgttt ttgctcaccc agaaacgctg gtgaaagtaa aagatgctga
     2341 agatcagttg ggtgcacgag tgggttacat cgaactggat ctcaacagcg gtaagatcct
     2401 tgagagtttt cgccccgaag aacgttttcc aatgatgagc acttttaaag ttctgctatg
     2461 tggcgcggta ttatcccgtg ttgacgccgg gcaagagcaa ctcggtcgcc gcatacacta
     2521 ttctcagaat gacttggttg agtactcacc agtcacagaa aagcatctta cggatggcat
     2581 gacagtaaga gaattatgca gtgctgccat aaccatgagt gataacactg cggccaactt
     2641 acttctgaca acgatcggag gaccgaagga gctaaccgct tttttgcaca acatggggga
     2701 tcatgtaact cgccttgatc gttgggaacc ggagctgaat gaagccatac caaacgacga
     2761 gcgtgacacc acgatgcctg cagcaatggc aacaacgttg cgcaaactat taactggcga
     2821 actacttact ctagcttccc ggcaacaatt aatagactgg atggaggcgg ataaagttgc
     2881 aggaccactt ctgcgctcgg cccttccggc tggctggttt attgctgata aatctggagc
     2941 cggtgagcgt gggtctcgcg gtatcattgc agcactgggg ccagatggta agccctcccg
     3001 tatcgtagtt atctacacga cggggagtca ggcaactatg gatgaacgaa atagacagat
     3061 cgctgagata ggtgcctcac tgattaagca ttggtaactg tcagaccaag tttactcata
     3121 tatactttag attgatttaa aacttcattt ttaatttaaa aggatctagg tgaagatcct
     3181 ttttgataat ctcatgacca aaatccctta acgtgagttt tcgttccact gagcgtcaga
     3241 ccccgtagaa aagatcaaag gatcttcttg agatcctttt tttctgcgcg taatctgctg
     3301 cttgcaaaca aaaaaaccac cgctaccagc ggtggtttgt ttgccggatc aagagctacc
     3361 aactcttttt ccgaaggtaa ctggcttcag cagagcgcag ataccaaata ctgtccttct
     3421 agtgtagccg tagttaggcc accacttcaa gaactctgta gcaccgccta catacctcgc
     3481 tctgctaatc ctgttaccag tggctgctgc cagtggcgat aagtcgtgtc ttaccgggtt
     3541 ggactcaaga cgatagttac cggataaggc gcagcggtcg ggctgaacgg ggggttcgtg
     3601 cacacagccc agcttggagc gaacgaccta caccgaactg agatacctac agcgtgagct
     3661 atgagaaagc gccacgcttc ccgaagggag aaaggcggac aggtatccgg taagcggcag
     3721 ggtcggaaca ggagagcgca cgagggagct tccaggggga aacgcctggt atctttatag
     3781 tcctgtcggg tttcgccacc tctgacttga gcgtcgattt ttgtgatgct cgtcaggggg
     3841 gcggagccta tggaaaaacg ccagcaacgc ggccttttta cggttcctgg ccttttgctg
     3901 gccttttgct cacatgttct ttcctgcgtt atcccctgat tctgtggata accgtattac
     3961 cgcctttgag tgagctgata ccgctcgccg cagccgaacg accgagcgca gcgagtcagt
     4021 gagcgaggaa gcggaagagc gcctgatgcg gtattttctc cttacgcatc tgtgcggtat
     4081 ttcacaccgc atatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc
     4141 cagtatacac tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa
     4201 cacccgctga cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg
     4261 tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga
     4321 ggcagctgcg gtaaagctca tcagcgtggt cgtgaagcga ttcacagatg tctgcctgtt
     4381 catccgcgtc cagctcgttg agtttctcca gaagcgttaa tgtctggctt ctgataaagc
     4441 gggccatgtt aagggcggtt ttttcctgtt tggtcactga tgcctccgtg taagggggat
     4501 ttctgttcat gggggtaatg ataccgatga aacgagagag gatgctcacg atacgggtta
     4561 ctgatgatga acatgcccgg ttactggaac gttgtgaggg taaacaactg gcggtatgga
     4621 tgcggcggga ccagagaaaa atcactcagg gtcaatgcca gcgcttcgtt aatacagatg
     4681 taggtgttcc acagggtagc cagcagcatc ctgcgatgca gatccggaac ataatggtgc
     4741 agggcgctga cttccgcgtt tccagacttt acgaaacacg gaaaccgaag accattcatg
     4801 ttgttgctca ggtcgcagac gttttgcagc agcagtcgct tcacgttcgc tcgcgtatcg
     4861 gtgattcatt ctgctaacca gtaaggcaac cccgccagcc tagccgggtc ctcaacgaca
     4921 ggagcacgat catgcgcacc cgtggccagg acccaacgct gcccgagatg cgccgcgtgc
     4981 ggctgctgga gatggcggac gcgatggata tgttctgcca agggttggtt tgcgcattca
     5041 cagttctccg caagaattga ttggctccaa ttcttggagt ggtgaatccg ttagcgaggt
     5101 gccgccggct tccattcagg tcgaggtggc ccggctccat gcaccgcgac gcaacgcggg
     5161 gaggcagaca aggtataggg cggcgcctac aatccatgcc aacccgttcc atgtgctcgc
     5221 cgaggcggca taaatcgccg tgacgatcag cggtccagtg atcgaagtta ggctggtaag
     5281 agccgcgagc gatccttgaa gctgtccctg atggtcgtca tctacctgcc tggacagcat
     5341 ggcctgcaac gcgggcatcc cgatgccgcc ggaagcgaga agaatcataa tggggaaggc
     5401 catccagcct cgcgtcgcga acgccagcaa gacgtagccc agcgcgtcgg ccgccatgcc
     5461 ggcgataatg gcctgcttct cgccgaaacg tttggtggcg ggaccagtga cgaaggcttg
     5521 agcgagggcg tgcaagattc cgaataccgc aagcgacagg ccgatcatcg tcgcgctcca
     5581 gcgaaagcgg tcctcgccga aaatgaccca gagcgctgcc ggcacctgtc ctacgagttg
     5641 catgataaag aagacagtca taagtgcggc gacgatagtc atgccccgcg cccaccggaa
     5701 ggagctgact gggttgaagg ctctcaaggg catcggtcga gatcccggtg cctaatgagt
     5761 gagctaactt acattaattg cgttgcgctc actgcccgct ttccagtcgg gaaacctgtc
     5821 gtgccagctg cattaatgaa tcggccaacg cgcggggaga ggcggtttgc gtattgggcg
     5881 ccagggtggt ttttcttttc accagtgaga cgggcaacag ctgattgccc ttcaccgcct
     5941 ggccctgaga gagttgcagc aagcggtcca cgctggtttg ccccagcagg cgaaaatcct
     6001 gtttgatggt ggttaacggc gggatataac atgagctgtc ttcggtatcg tcgtatccca
     6061 ctaccgagat atccgcacca acgcgcagcc cggactcggt aatggcgcgc attgcgccca
     6121 gcgccatctg atcgttggca accagcatcg cagtgggaac gatgccctca ttcagcattt
     6181 gcatggtttg ttgaaaaccg gacatggcac tccagtcgcc ttcccgttcc gctatcggct
     6241 gaatttgatt gcgagtgaga tatttatgcc agccagccag acgcagacgc gccgagacag
     6301 aacttaatgg gcccgctaac agcgcgattt gctggtgacc caatgcgacc agatgctcca
     6361 cgcccagtcg cgtaccgtct tcatgggaga aaataatact gttgatgggt gtctggtcag
     6421 agacatcaag aaataacgcc ggaacattag tgcaggcagc ttccacagca atggcatcct
     6481 ggtcatccag cggatagtta atgatcagcc cactgacgcg ttgcgcgaga agattgtgca
     6541 ccgccgcttt acaggcttcg acgccgcttc gttctaccat cgacaccacc acgctggcac
     6601 ccagttgatc ggcgcgagat ttaatcgccg cgacaatttg cgacggcgcg tgcagggcca
     6661 gactggaggt ggcaacgcca atcagcaacg actgtttgcc cgccagttgt tgtgccacgc
     6721 ggttgggaat gtaattcagc tccgccatcg ccgcttccac tttttcccgc gttttcgcag
     6781 aaacgtggct ggcctggttc accacgcggg aaacggtctg ataagagaca ccggcatact
     6841 ctgcgacatc gtataacgtt actggtttca cattcaccac cctgaattga ctctcttccg
     6901 ggcgctatca tgccataccg cgaaaggttt tgcgccattc gatggtgtcc gggatctcga
     6961 cgctctccct tatgcgactc ctgcattagg aagcagccca gtagtaggtt gaggccgttg
     7021 agcaccgccg ccgcaaggaa tggtgcatgc aaggagatgg cgcccaacag tcccccggcc
     7081 acggggcctg ccaccatacc cacgccgaaa caagcgctca tgagcccgaa gtggcgagcc
     7141 cgatcttccc catcggtgat gtcggcgata taggcgccag caaccgcacc tgtggcgccg
     7201 gtgatgccgg ccacgatgcg tccggcgtag aggatcga
//
