LOCUS       sequence-12934-e        7287 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  synthetic circular DNA
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 7287)
  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 7287)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 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-AP
FEATURES             Location/Qualifiers
     source          1..7287
                     /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"
     CDS             1706..1750
                     /codon_start=1
                     /product="peptide tag that allows for enzymatic 
                     biotinylation"
                     /label=AviTag(TM)
                     /translation="GLNDIFEAQKIEWHE"
     primer_bind     complement(1804..1822)
                     /label=T7 Term
                     /note="T7 terminator, reverse primer"
     terminator      1818..1865
                     /label=T7 terminator
                     /note="transcription terminator for bacteriophage T7 RNA 
                     polymerase"
     promoter        complement(join(2040..2045,2046..2062,2063..2068))
                     /label=tet promoter
                     /note="E. coli promoter for tetracycline efflux protein 
                     gene"
     primer_bind     complement(2095..2113)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        2181..2285
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(2286..2354,2355..3146)
                     /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(2504..2523)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      3317..3905
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3806..3825
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     4059..4076
                     /label=L4440
                     /note="L4440 vector, forward primer"
     misc_feature    4091..4233
                     /label=bom
                     /note="basis of mobility region from pBR322"
     primer_bind     complement(4159..4178)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     4319..4341
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     CDS             complement(4335..4526)
                     /codon_start=1
                     /gene="rop"
                     /product="Rop protein, which maintains plasmids at low copy
                     number"
                     /label=rop
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDHA
                     DELYRSCLARFGDDGENL"
     CDS             complement(5838..6920)
                     /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     6882..6901
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        complement(6921..6998)
                     /gene="lacI"
                     /label=lacI promoter
     primer_bind     7204..7223
                     /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 cgaacattgg 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 gcgcaaccgc tttgcgaaaa ttctgggcgt gtttggcctg tggattccgc tggtggtggc
      421 gatttattat gtgtatattg aaagctggac cctgggcttt gcgattaaat ttctggtggg
      481 cctggtgccg gaaccgccgc cgaacgcgac agatccggat 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 catcatggac tcgaggggct taatgatatc tttgaagctc agaaaattga
     1741 atggcatgag tgaggatccg gctgctaaca aagcccgaaa ggaagctgag ttggctgctg
     1801 ccaccgctga gcaataacta gcataacccc ttggggcctc taaacgggtc ttgaggggtt
     1861 ttttgctgaa aggaggaact atatccggat atcccgcaag aggcccggca gtaccggcat
     1921 aaccaagcct atgcctacag catccagggt gacggtgccg aggatgacga tgagcgcatt
     1981 gttagatttc atacacggtg cctgactgcg ttagcaattt aactgtgata aactaccgca
     2041 ttaaagctta tcgatgataa gctgtcaaac atgagaattc ttgaagacga aagggcctcg
     2101 tgatacgcct atttttatag gttaatgtca tgataataat ggtttcttag acgtcaggtg
     2161 gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt atttttctaa atacattcaa
     2221 atatgtatcc gctcatgaga caataaccct gataaatgct tcaataatat tgaaaaagga
     2281 agagtatgag tattcaacat ttccgtgtcg cccttattcc cttttttgcg gcattttgcc
     2341 ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa agatgctgaa gatcagttgg
     2401 gtgcacgagt gggttacatc gaactggatc tcaacagcgg taagatcctt gagagttttc
     2461 gccccgaaga acgttttcca atgatgagca cttttaaagt tctgctatgt ggcgcggtat
     2521 tatcccgtgt tgacgccggg caagagcaac tcggtcgccg catacactat tctcagaatg
     2581 acttggttga gtactcacca gtcacagaaa agcatcttac ggatggcatg acagtaagag
     2641 aattatgcag tgctgccata accatgagtg ataacactgc ggccaactta cttctgacaa
     2701 cgatcggagg accgaaggag ctaaccgctt ttttgcacaa catgggggat catgtaactc
     2761 gccttgatcg ttgggaaccg gagctgaatg aagccatacc aaacgacgag cgtgacacca
     2821 cgatgcctgc agcaatggca acaacgttgc gcaaactatt aactggcgaa ctacttactc
     2881 tagcttcccg gcaacaatta atagactgga tggaggcgga taaagttgca ggaccacttc
     2941 tgcgctcggc ccttccggct ggctggttta ttgctgataa atctggagcc ggtgagcgtg
     3001 ggtctcgcgg tatcattgca gcactggggc cagatggtaa gccctcccgt atcgtagtta
     3061 tctacacgac ggggagtcag gcaactatgg atgaacgaaa tagacagatc gctgagatag
     3121 gtgcctcact gattaagcat tggtaactgt cagaccaagt ttactcatat atactttaga
     3181 ttgatttaaa acttcatttt taatttaaaa ggatctaggt gaagatcctt tttgataatc
     3241 tcatgaccaa aatcccttaa cgtgagtttt cgttccactg agcgtcagac cccgtagaaa
     3301 agatcaaagg atcttcttga gatccttttt ttctgcgcgt aatctgctgc ttgcaaacaa
     3361 aaaaaccacc gctaccagcg gtggtttgtt tgccggatca agagctacca actctttttc
     3421 cgaaggtaac tggcttcagc agagcgcaga taccaaatac tgtccttcta gtgtagccgt
     3481 agttaggcca ccacttcaag aactctgtag caccgcctac atacctcgct ctgctaatcc
     3541 tgttaccagt ggctgctgcc agtggcgata agtcgtgtct taccgggttg gactcaagac
     3601 gatagttacc ggataaggcg cagcggtcgg gctgaacggg gggttcgtgc acacagccca
     3661 gcttggagcg aacgacctac accgaactga gatacctaca gcgtgagcta tgagaaagcg
     3721 ccacgcttcc cgaagggaga aaggcggaca ggtatccggt aagcggcagg gtcggaacag
     3781 gagagcgcac gagggagctt ccagggggaa acgcctggta tctttatagt cctgtcgggt
     3841 ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc gtcagggggg cggagcctat
     3901 ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc cttttgctgg ccttttgctc
     3961 acatgttctt tcctgcgtta tcccctgatt ctgtggataa ccgtattacc gcctttgagt
     4021 gagctgatac cgctcgccgc agccgaacga ccgagcgcag cgagtcagtg agcgaggaag
     4081 cggaagagcg cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca
     4141 tatatggtgc actctcagta caatctgctc tgatgccgca tagttaagcc agtatacact
     4201 ccgctatcgc tacgtgactg ggtcatggct gcgccccgac acccgccaac acccgctgac
     4261 gcgccctgac gggcttgtct gctcccggca tccgcttaca gacaagctgt gaccgtctcc
     4321 gggagctgca tgtgtcagag gttttcaccg tcatcaccga aacgcgcgag gcagctgcgg
     4381 taaagctcat cagcgtggtc gtgaagcgat tcacagatgt ctgcctgttc atccgcgtcc
     4441 agctcgttga gtttctccag aagcgttaat gtctggcttc tgataaagcg ggccatgtta
     4501 agggcggttt tttcctgttt ggtcactgat gcctccgtgt aagggggatt tctgttcatg
     4561 ggggtaatga taccgatgaa acgagagagg atgctcacga tacgggttac tgatgatgaa
     4621 catgcccggt tactggaacg ttgtgagggt aaacaactgg cggtatggat gcggcgggac
     4681 cagagaaaaa tcactcaggg tcaatgccag cgcttcgtta atacagatgt aggtgttcca
     4741 cagggtagcc agcagcatcc tgcgatgcag atccggaaca taatggtgca gggcgctgac
     4801 ttccgcgttt ccagacttta cgaaacacgg aaaccgaaga ccattcatgt tgttgctcag
     4861 gtcgcagacg ttttgcagca gcagtcgctt cacgttcgct cgcgtatcgg tgattcattc
     4921 tgctaaccag taaggcaacc ccgccagcct agccgggtcc tcaacgacag gagcacgatc
     4981 atgcgcaccc gtggccagga cccaacgctg cccgagatgc gccgcgtgcg gctgctggag
     5041 atggcggacg cgatggatat gttctgccaa gggttggttt gcgcattcac agttctccgc
     5101 aagaattgat tggctccaat tcttggagtg gtgaatccgt tagcgaggtg ccgccggctt
     5161 ccattcaggt cgaggtggcc cggctccatg caccgcgacg caacgcgggg aggcagacaa
     5221 ggtatagggc ggcgcctaca atccatgcca acccgttcca tgtgctcgcc gaggcggcat
     5281 aaatcgccgt gacgatcagc ggtccagtga tcgaagttag gctggtaaga gccgcgagcg
     5341 atccttgaag ctgtccctga tggtcgtcat ctacctgcct ggacagcatg gcctgcaacg
     5401 cgggcatccc gatgccgccg gaagcgagaa gaatcataat ggggaaggcc atccagcctc
     5461 gcgtcgcgaa cgccagcaag acgtagccca gcgcgtcggc cgccatgccg gcgataatgg
     5521 cctgcttctc gccgaaacgt ttggtggcgg gaccagtgac gaaggcttga gcgagggcgt
     5581 gcaagattcc gaataccgca agcgacaggc cgatcatcgt cgcgctccag cgaaagcggt
     5641 cctcgccgaa aatgacccag agcgctgccg gcacctgtcc tacgagttgc atgataaaga
     5701 agacagtcat aagtgcggcg acgatagtca tgccccgcgc ccaccggaag gagctgactg
     5761 ggttgaaggc tctcaagggc atcggtcgag atcccggtgc ctaatgagtg agctaactta
     5821 cattaattgc gttgcgctca ctgcccgctt tccagtcggg aaacctgtcg tgccagctgc
     5881 attaatgaat cggccaacgc gcggggagag gcggtttgcg tattgggcgc cagggtggtt
     5941 tttcttttca ccagtgagac gggcaacagc tgattgccct tcaccgcctg gccctgagag
     6001 agttgcagca agcggtccac gctggtttgc cccagcaggc gaaaatcctg tttgatggtg
     6061 gttaacggcg ggatataaca tgagctgtct tcggtatcgt cgtatcccac taccgagata
     6121 tccgcaccaa cgcgcagccc ggactcggta atggcgcgca ttgcgcccag cgccatctga
     6181 tcgttggcaa ccagcatcgc agtgggaacg atgccctcat tcagcatttg catggtttgt
     6241 tgaaaaccgg acatggcact ccagtcgcct tcccgttccg ctatcggctg aatttgattg
     6301 cgagtgagat atttatgcca gccagccaga cgcagacgcg ccgagacaga acttaatggg
     6361 cccgctaaca gcgcgatttg ctggtgaccc aatgcgacca gatgctccac gcccagtcgc
     6421 gtaccgtctt catgggagaa aataatactg ttgatgggtg tctggtcaga gacatcaaga
     6481 aataacgccg gaacattagt gcaggcagct tccacagcaa tggcatcctg gtcatccagc
     6541 ggatagttaa tgatcagccc actgacgcgt tgcgcgagaa gattgtgcac cgccgcttta
     6601 caggcttcga cgccgcttcg ttctaccatc gacaccacca cgctggcacc cagttgatcg
     6661 gcgcgagatt taatcgccgc gacaatttgc gacggcgcgt gcagggccag actggaggtg
     6721 gcaacgccaa tcagcaacga ctgtttgccc gccagttgtt gtgccacgcg gttgggaatg
     6781 taattcagct ccgccatcgc cgcttccact ttttcccgcg ttttcgcaga aacgtggctg
     6841 gcctggttca ccacgcggga aacggtctga taagagacac cggcatactc tgcgacatcg
     6901 tataacgtta ctggtttcac attcaccacc ctgaattgac tctcttccgg gcgctatcat
     6961 gccataccgc gaaaggtttt gcgccattcg atggtgtccg ggatctcgac gctctccctt
     7021 atgcgactcc tgcattagga agcagcccag tagtaggttg aggccgttga gcaccgccgc
     7081 cgcaaggaat ggtgcatgca aggagatggc gcccaacagt cccccggcca cggggcctgc
     7141 caccataccc acgccgaaac aagcgctcat gagcccgaag tggcgagccc gatcttcccc
     7201 atcggtgatg tcggcgatat aggcgccagc aaccgcacct gtggcgccgg tgatgccggc
     7261 cacgatgcgt ccggcgtaga ggatcga
//
