LOCUS       sequence-35859-e        8504 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 8504)
  AUTHORS   Petzold AM, Balciunas D, Sivasubbu S, Clark KJ, Bedell VM, Westcot 
            SE, Myers SR, Moulder GL, Thomas MJ, Ekker SC
  TITLE     Nicotine response genetics in the zebrafish.
  JOURNAL   Proc Natl Acad Sci U S A. 2009 Nov 3;106(44):18662-7. Epub 2009 Oct 
            26.
  PUBMED    19858493
REFERENCE   2  (bases 1 to 8504)
  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: "Proc Natl 
            Acad Sci U S A."; date: "2009-11-3"; volume: "106(44)"; pages: 
            "18662-7. Epub 2009 Oct 26"
COMMENT     Sequence Label: pGBT-PX
FEATURES             Location/Qualifiers
     source          1..8504
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    join(2..13,14..176)
                     /label=Tol2 right end
                     /note="right end of the Tol2 transposon from the medaka 
                     fish Oryzias latipes (Kawakami, 2007)"
     protein_bind    complement(227..260)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     primer_bind     1739..1759
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        1741..1759
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     CDS             4329..5042
                     /codon_start=1
                     /product="S65T variant of Aequorea victoria green 
                     fluorescent protein (Heim et al., 1995)"
                     /label=GFP (S65T)
                     /note="excitable with blue light"
                     /translation="MSKGEELFTGVVPILVELNGDVNGHKFPVSGEGEGDATYGKLTLK
                     FICTTGKLPVPWPTLVTTLAYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDG
                     NYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKV
                     NFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLE
                     FVTAAGITHGMDELYK"
     primer_bind     4982..5002
                     /label=GFP-F
                     /note="GFP, forward primer. Does NOT anneal to EGFP"
     protein_bind    complement(5254..5287)
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     misc_feature    join(5366..5553,5554..5565)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(5577..5596)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(5578..5596)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(5603..5620)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(5603..5619)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(5612..5634)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      5760..6215
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(5847..5866)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     6057..6078
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        6293..6397
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(6398..6466,6467..7258)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRIDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(6616..6635)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      7429..8017
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     7918..7937
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     8171..8188
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    8305..8326
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(8341..8346,8347..8364,8365..8371)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    8379..8395
                     /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     8384..8406
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     8403..8419
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     8403..8419
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        8437..8455
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     8437..8454
                     /label=SP6
                     /note="SP6 promoter, forward primer"
ORIGIN
        1 gcagaggtgt aaaaagtact caaaaatttt actcaagtga aagtacaagt acttagggaa
       61 aattttactc aattaaaagt aaaagtatct ggctagaatc ttacttgagt aaaagtaaaa
      121 aagtactcca ttaaaattgt acttgagtat taaggaagta aaagtaaaag caagaaagaa
      181 aactagagat tcttgtttaa gctcatggta gggataacag ggtaatataa cttcgtatag
      241 catacattat acgaagttat cgttaattaa gaagttccta tactttctag agaataggaa
      301 gatctccgga cgaaacagga agctgactcc acatggtcac atgctcactg aagtgttgac
      361 ttccctgaca gctgtgcact ttctaaaccg gttttctcat tcatttacag ttcagccgat
      421 gatgaaattg ccgcactggt tgttagcaac gtagccggtg tgtgaaagat ggattcgcgg
      481 gaattccgaa gctagaacta gtaattaagt ctcagccacc gttaactgaa catgtcaaaa
      541 cctgtggaga ctgttgagat ttgatgttct gaaaagataa agcctataaa taaaatgttg
      601 cccaaatttc ctgcctgatg tttttctttg tctttgctac atggctttgc tgctcggatc
      661 ggctcactct gtgtatgcca cgttcacttt gtactctcct tctcacggta ggtttattat
      721 ttttagatgt gcagttagtt tctgtgaaat aacacaccac acactgatat tgtctgtgca
      781 ttgacttggt gagtgcacat tgtttttgat cttgacatat ttatatttga ttgatcaggt
      841 gaactgtgtg aatctaaagt gctccataca gatgttctgc attgaaaata ttctcatttt
      901 attagtggaa gtgagtgtat gccacatcca atcaatttca gcaaacaccc cagtatgatt
      961 taatgcaaaa aaatgaaggt atcaaacacg cattactact ttgcagttaa atatttaaca
     1021 tttattccaa cacgaaaaaa agcagtaaat aacactttga caaacacgtc aggacatctt
     1081 atttttgtca ccctcacagg caatttagta taatatatta tatatatata tatatcatat
     1141 aataatattc agtataatat atatatatat atcatattat aatattcagt ataatataaa
     1201 acacaaacac atatatgtat aatataatat aacattttta tttattgaga tgcctctatg
     1261 gaccgtgtta taagaagtaa agatcaggag aagtaaacat gaagtgtaat tatgaatact
     1321 gatgttaaat taagctatga tgagttttca ctgttaattt accatctcaa ttaaatgttg
     1381 atgcctccat gaccaagtta agcagatgag actgagacaa ctgtagaaga caagatgttc
     1441 actttgctga atatagctgg cttgacagtt atctatgact ctataaatat atatatattt
     1501 ttttttttat aaaatgattt atttataact atatatccat ttctcagaca ggtgcttcat
     1561 atccctcact cccgtagctg tccatgctgg atctgtcccc gttgttttta aaaagctaaa
     1621 taagttatta acatgactgc atccagcgag ccaaacctgt ctggtgtaca gctaccagag
     1681 aagcttgaga tcctaggaag ttcctatact ttctagagaa taggaatcgc gagtgctagc
     1741 aattaaccct cactaaaggg aacaaaagct tttagacctt cttacttttg gggattatat
     1801 aagtattttc tcaataaata tctcatatct tactgtggtt taactgctga atctaaaatt
     1861 ttaatacaaa agtagttata tttgttgtac attgtaaact ataacttaac ttcagtttca
     1921 gagaaactca tgtgctcaaa atgtaaaaaa agtttcctgt taaatatttt gtaaatgtat
     1981 tgaagacaaa ataagaaaaa aaaaaatata agccactaaa tcacactgtc cttggtatca
     2041 gcaagagatt ctgacataat cagctgtttt tgtttattac tgccattgaa ggccatgtgc
     2101 attagtccca agttacacat taaaaagtca catgtagctt accaacatca gtgctgttca
     2161 agcacagcct catctactat tcaaactgtg gcaccatcta aaatatgcca gaattttttt
     2221 atttaatgaa tttgaccctg aaatatgtat taatatcact cctgtgattt ttttgtaatc
     2281 agcttacaat tacaggaatg caagcctgat tcattacaag tttcactaca ctttctctga
     2341 caacatcacc tactgaactc agaccagcta gttgctcctt aagtatacaa tcatgtcagt
     2401 aatcctcatt tcaatgaaaa atacccgtat tgtacttggt acttggtaga taaccacaga
     2461 gcagtattat gccattattg tgaatacaat aagaggtaaa tgacctacag agctgctgct
     2521 gctgttgtgt tagattgtaa acacagcaca ggatcaagga ggtgtccatc actatgacca
     2581 atactagcac tttgcacagg ctctttgaaa ggctgaaaag agccttattg gcgttatcac
     2641 aacaaaatac gcaaatacgg aaaacaacgt attgaacttc gcaaacaaaa aacagcgatt
     2701 ttgatgaaaa tcgcttaggc cttgctcttc aaacaatcca gcttctcctt ctttcactct
     2761 caagttgcaa gaagcaagtg tagcaatgtg cacgcgacag ccgggtgtgt gacgctggac
     2821 caatcagagc gcagagctcc gaaagtttac cttttatggc tagagccggc atctgccgtc
     2881 atataaaaga gcgcgcccag cgtctcagcc tcactttgag ctcctccaca cgcagctagt
     2941 gcggaatatc atctgcctgt aacccattct ctaaagtcga caaacccccc caaacctaag
     3001 gtgagttgat ctttaagctt tttacatttt cagctcgcat atatcaattc gaacgtttaa
     3061 ttagaatgtt taaataaagc tagattaaat gattaggctc agttaccggt cttttttttc
     3121 tcatttacgt gcgaactctg cttaaactct agttattctt tattaatatg tggttatttt
     3181 tatatatgta tgttatcata actgtactgg ctatgtcagg tggtaatgac tgtaacgtta
     3241 cgttactcgt tgtaggcacg acattgaatg ggccggtgtt gaaataagtc ttcaacccct
     3301 tttaacctca aaatgtgctc tggttaacaa ggattttaac agctatcagt atgactgtgc
     3361 ggttttaaag ccgttagtga ggcacgttgc acacttgatg gatggccgga atgggaagtt
     3421 ctttatgcag gcagtgctgc agcagggtgt gacctacttt agctaacgtt agccggctaa
     3481 ccagcattca tctgccggta acttgagtct aatattctct atgtgatatc gaagtgatca
     3541 aagacacgtc tgttagctca ctttaaccaa ctgtagtgaa aaatagcgca gtgtgcagcc
     3601 cttcaagtct ttcatttagg ctgattattc aatcatttta ttaactatta acgcgttact
     3661 aaacgtaagg taacgtagtc agtttttaat aactggtgaa aagtactggt tgggtttaaa
     3721 tggtgactta taattgtgtt ggagggggaa acctttttga taaaggctat ataatctcaa
     3781 atgaatgggc tgaggatggt gttcacaggt gctttagtga agtccgctcg tgaagagtcg
     3841 ctgaagtgac tgcagatctg tagcgcatgc gttttggcag acggccgttg aaattcggtt
     3901 gagtaattga taccaggtga ggctagagga tgtagaaatt catttgtgta gaatttaggg
     3961 agtggcctgg cgtgatgaat gtcgaaatcc gttccttttt actgaaccct atgtctctgc
     4021 tgagtgccac accgccggca caaagcgtct caaaccattg ccttttatgg taataatgag
     4081 aatgcagagg gacttccttt gtctggcaca tctgaggcgc gcattgtcac actagcaccc
     4141 actagcggtc agactgcaga caaacaggaa gctgactcca catggtcaca tgctcactga
     4201 agtgttgact tccctgacag ctgtgcactt tctaaaccgg ttttctcatt catttacagt
     4261 tcagccggaa ttcacctgca ggtggatcca tctagaacta ctagaggatc cccgggtacc
     4321 ggtagaaaat gagtaaagga gaagaacttt tcactggagt tgtcccaatt cttgttgaat
     4381 taaatggtga tgttaatggg cacaaatttc ctgtcagtgg agagggtgaa ggtgatgcaa
     4441 catacggaaa acttaccctt aaatttattt gcactactgg aaaactacct gttccatggc
     4501 caacacttgt cactactctc gcgtatggtc ttcaatgctt tgcgagatac ccagatcata
     4561 tgaaacagca tgactttttc aagagtgcca tgcccgaagg ttatgtacag gaaagaacta
     4621 tatttttcaa agatgacggg aactacaaga cacgtgctga agtcaagttt gaaggtgata
     4681 cccttgttaa tagaatcgag ttaaaaggta ttgattttaa agaagatgga aacattcttg
     4741 gacacaaatt ggaatacaac tataactcac acaatgtata catcatggca gacaaacaaa
     4801 agaatggaat caaagttaac ttcaaaatta gacacaacat tgaagatgga agcgttcaac
     4861 tagcagacca ttatcaacaa aatactccaa ttggcgatgg ccctgtcctt ttaccagaca
     4921 accattacct gtccacacaa tctgcccttt cgaaagatcc caacgaaaag agagaccaca
     4981 tggtccttct tgagtttgta acagctgctg ggattacaca tggcatggat gaactataca
     5041 aataaatgtc cagacctgca ggaattcgat atcaagctta tcgatgaaca aaccccccca
     5101 aacctaaggt gagttgatct ttaagctttt tacattttca gctcgcatat atcaattcga
     5161 acgtttaatt agaatgttta aataaagcta gattaaatga ttaggctcag ttaccggtct
     5221 tttttttctc atttacacta gctaagcatg ctgataactt cgtatagcat acattatacg
     5281 aagttattac cctgttatcc ctacaaatta aactgggcat cagcgcaatt caattggttt
     5341 ggtaatagca agggaaaata gaatgaagtg atctccaaaa aataagtact ttttgactgt
     5401 aaataaaatt gtaaggagta aaaagtactt ttttttctaa aaaaatgtaa ttaagtaaaa
     5461 gtaaaagtat tgatttttaa ttgtactcaa gtaaagtaaa aatccccaaa aataatactt
     5521 aagtacagta atcaagtaaa attactcaag tactttacac ctctgggccc aattcgccct
     5581 atagtgagtc gtattacaat tcactggccg tcgttttaca acgtcgtgac tgggaaaacc
     5641 ctggcgttac ccaacttaat cgccttgcag cacatccccc tttcgccagc tggcgtaata
     5701 gcgaagaggc ccgcaccgat cgcccttccc aacagttgcg cagcctgaat ggcgaatgga
     5761 cgcgccctgt agcggcgcat taagcgcggc gggtgtggtg gttacgcgca gcgtgaccgc
     5821 tacacttgcc agcgccctag cgcccgctcc tttcgctttc ttcccttcct ttctcgccac
     5881 gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt tccgatttag
     5941 tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac gtagtgggcc
     6001 atcgccctga tagacggttt ttcgcccttt gacgttggag tccacgttct ttaatagtgg
     6061 actcttgttc caaactggaa caacactcaa ccctatctcg gtctattctt ttgatttata
     6121 agggattttg ccgatttcgg cctattggtt aaaaaatgag ctgatttaac aaaaatttaa
     6181 cgcgaatttt aacaaaatat taacgcttac aatttcctga tgcggtattt tctccttacg
     6241 catctgtgcg gtatttcaca ccgcatcagg tggcactttt cggggaaatg tgcgcggaac
     6301 ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga gacaataacc
     6361 ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac atttccgtgt
     6421 cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc cagaaacgct
     6481 ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca tcgaactgga
     6541 tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc caatgatgag
     6601 cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg ggcaagagca
     6661 actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac cagtcacaga
     6721 aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca taaccatgag
     6781 tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg agctaaccgc
     6841 ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac cggagctgaa
     6901 tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg caacaacgtt
     6961 gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat taatagactg
     7021 gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg ctggctggtt
     7081 tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg cagcactggg
     7141 gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc aggcaactat
     7201 ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc attggtaact
     7261 gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt tttaatttaa
     7321 aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt aacgtgagtt
     7381 ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt
     7441 ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg
     7501 tttgccggat caagagctac caactctttt tccgaaggta actggcttca gcagagcgca
     7561 gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt
     7621 agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga
     7681 taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc
     7741 gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct acaccgaact
     7801 gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga
     7861 caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg
     7921 aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg agcgtcgatt
     7981 tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg cggccttttt
     8041 acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt tatcccctga
     8101 ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc gcagccgaac
     8161 gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac gcaaaccgcc
     8221 tctccccgcg cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa
     8281 agcgggcagt gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc
     8341 tttacacttt atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca
     8401 cacaggaaac agctatgacc atgattacgc caagctattt aggtgacact atagaatact
     8461 caagctatgc atccaacgcg ttgggagctc tcccatatgg tcga
//
