LOCUS       sequence-35861-e        5469 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 5469)
  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 5469)
  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-R15
FEATURES             Location/Qualifiers
     source          1..5469
                     /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    221..254
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     CDS             428..1102
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     CDS             428..1102
                     /codon_start=1
                     /product="monomeric derivative of DsRed (Campbell et al., 
                     2002)"
                     /label=mRFP1
                     /note="mammalian codon-optimized"
                     /translation="ASSEDVIKEFMRFKVRMEGSVNGHEFEIEGEGEGRPYEGTQTAKL
                     KVTKGGPLPFAWDILSPQFQYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVV
                     TVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASTERMYPEDGALKGEIKMR
                     LKLKDGGHYDAEVKTTYMAKKPVQLPGAYKTDIKLDITSHNEDYTIVEQYERAEGRHST
                     GA"
     primer_bind     complement(555..573)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     823..842
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     1017..1040
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     protein_bind    2213..2246
                     /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(2331..2518,2519..2530)
                     /label=Tol2 left end
                     /note="left end of the Tol2 transposon from the medaka fish
                     Oryzias latipes (Kawakami, 2007)"
     primer_bind     complement(2542..2561)
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        complement(2543..2561)
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     complement(2568..2585)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(2568..2584)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2577..2599)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     rep_origin      2725..3180
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(2812..2831)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3022..3043
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     promoter        3258..3362
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(3363..3431,3432..4223)
                     /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(3581..3600)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      4394..4982
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     4883..4902
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     5136..5153
                     /label=L4440
                     /note="L4440 vector, forward primer"
     protein_bind    5270..5291
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     promoter        join(5306..5311,5312..5329,5330..5336)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    5344..5360
                     /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     5349..5371
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     primer_bind     5368..5384
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     5368..5384
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     promoter        5402..5420
                     /label=SP6 promoter
                     /note="promoter for bacteriophage SP6 RNA polymerase"
     primer_bind     5402..5419
                     /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 gctgtaggga taacagggta ataacttcgt ataatgtatg
      241 ctatacgaag ttatcagcat gccacccact agcggtcaga ctgcagattg cagcacgaaa
      301 caggaagctg actccacatg gtcacatgct cactgaagtg ttgacttccc tgacagctgt
      361 gcactttcta aaccggtttt ctcattcatt tacagttcag ccggaattca cccgggtacc
      421 ggaaggtgcc tcctccgagg acgtcatcaa ggagttcatg cgcttcaagg tgcgcatgga
      481 gggctccgtg aacggccacg agttcgagat cgagggcgag ggcgagggcc gcccctacga
      541 gggcacccag accgccaagc tgaaggtgac caagggcggc cccctgccct tcgcctggga
      601 catcctgtcc cctcagttcc agtacggctc caaggcctac gtgaagcacc ccgccgacat
      661 ccccgactac ttgaagctgt ccttccccga gggcttcaag tgggagcgcg tgatgaactt
      721 cgaggacggc ggcgtggtga ccgtgaccca ggactcctcc ctgcaggacg gcgagttcat
      781 ctacaaggtg aagctgcgcg gcaccaactt cccctccgac ggccccgtaa tgcagaagaa
      841 gaccatgggc tgggaggcct ccaccgagcg gatgtacccc gaggacggcg ccctgaaggg
      901 cgagatcaag atgaggctga agctgaagga cggcggccac tacgacgccg aggtcaagac
      961 cacctacatg gccaagaagc ccgtgcagct gcccggcgcc tacaagaccg acatcaagct
     1021 ggacatcacc tcccacaacg aggactacac catcgtggaa cagtacgagc gcgccgaggg
     1081 ccgccactcc accggcgcct aagaaatcga tacggactgt taccacttca cgccgactca
     1141 aactgcgcag agaaacttca aacgacaaca ttggcatggc ttttgttatt tttggcgctt
     1201 gactcaggat ctaaaaactg gaacggtgaa ggtgacggca atgttttttg gcaaataagc
     1261 atccccgaag ttctacaatg catctgagga ctccatgttt tgttttcttt agtcattcca
     1321 aatgtttgtt aaatgcattg ttccgaaact tatttgcctc tatgaaggct gcccagtaat
     1381 tgggagcata cttaacattg tagtattgta tgtaaattat gtaacaaaac aatgtctggg
     1441 tttttgtact tttcagcctt aaaatcttgg gtttattttt ttttctttgt tccaaaaaaa
     1501 cgaagcttta ccattcaaga tgtaaaggtt tccattcccc ctgggcatat tgtaaaagct
     1561 gtgtggaacg tggcggtgcc agacatttgg tggggccaac ctgtacactg actaattcaa
     1621 ttccaataaa agtgcacatg taagacatcc tacccctgtc tcatttcctg tcttggctct
     1681 gagtggcttc aataaaaatg gcttttgtga agttgcacca tgttaattac atttcagcac
     1741 tttcacacca ggtttttgac taatttgaat ttatcgaagt ttgacctttt tttttttttt
     1801 tccttcaagc taatttgagg ttatcaaacc atttcattgg aggaaactta atgtttaaaa
     1861 gcataaattc gaaacatgat tcatttattt aactgcttaa tggtgaaaca aacttcaagt
     1921 aatacaccaa gtgttccctc atatgaaggt agctaactgc aaacggcctt aactttccat
     1981 cttaagttga gctacataaa ggctttaaca ggaaggaaga gccagtttaa aagtcaaacc
     2041 accatgactg atataaattc ttgtccatat aaccttgcgt ttcacacaat atagctatct
     2101 actgctaaat aaattatcgc gattcctatt ctctagaaag tataggaact tcctaggagc
     2161 gctccggaga tcttcctatt ctctagaaag tataggaact tcttaattaa cgataacttc
     2221 gtataatgta tgctatacga agttatatta ccctgttatc cctaccatga aattaaactg
     2281 ggcatcagcg caattcaatt ggtttggtaa tagcaaggga aaatagaatg aagtgatctc
     2341 caaaaaataa gtactttttg actgtaaata aaattgtaag gagtaaaaag tacttttttt
     2401 tctaaaaaaa tgtaattaag taaaagtaaa agtattgatt tttaattgta ctcaagtaaa
     2461 gtaaaaatcc ccaaaaataa tacttaagta cagtaatcaa gtaaaattac tcaagtactt
     2521 tacacctctg ggcccaattc gccctatagt gagtcgtatt acaattcact ggccgtcgtt
     2581 ttacaacgtc gtgactggga aaaccctggc gttacccaac ttaatcgcct tgcagcacat
     2641 ccccctttcg ccagctggcg taatagcgaa gaggcccgca ccgatcgccc ttcccaacag
     2701 ttgcgcagcc tgaatggcga atggacgcgc cctgtagcgg cgcattaagc gcggcgggtg
     2761 tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc gctcctttcg
     2821 ctttcttccc ttcctttctc gccacgttcg ccggctttcc ccgtcaagct ctaaatcggg
     2881 ggctcccttt agggttccga tttagtgctt tacggcacct cgaccccaaa aaacttgatt
     2941 agggtgatgg ttcacgtagt gggccatcgc cctgatagac ggtttttcgc cctttgacgt
     3001 tggagtccac gttctttaat agtggactct tgttccaaac tggaacaaca ctcaacccta
     3061 tctcggtcta ttcttttgat ttataaggga ttttgccgat ttcggcctat tggttaaaaa
     3121 atgagctgat ttaacaaaaa tttaacgcga attttaacaa aatattaacg cttacaattt
     3181 cctgatgcgg tattttctcc ttacgcatct gtgcggtatt tcacaccgca tcaggtggca
     3241 cttttcgggg aaatgtgcgc ggaaccccta tttgtttatt tttctaaata cattcaaata
     3301 tgtatccgct catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga
     3361 gtatgagtat tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc
     3421 ctgtttttgc tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg
     3481 cacgagtggg ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc
     3541 ccgaagaacg ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat
     3601 cccgtattga cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact
     3661 tggttgagta ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat
     3721 tatgcagtgc tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga
     3781 tcggaggacc gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc
     3841 ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga
     3901 tgcctgtagc aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag
     3961 cttcccggca acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc
     4021 gctcggccct tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt
     4081 ctcgcggtat cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct
     4141 acacgacggg gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg
     4201 cctcactgat taagcattgg taactgtcag accaagttta ctcatatata ctttagattg
     4261 atttaaaact tcatttttaa tttaaaagga tctaggtgaa gatccttttt gataatctca
     4321 tgaccaaaat cccttaacgt gagttttcgt tccactgagc gtcagacccc gtagaaaaga
     4381 tcaaaggatc ttcttgagat cctttttttc tgcgcgtaat ctgctgcttg caaacaaaaa
     4441 aaccaccgct accagcggtg gtttgtttgc cggatcaaga gctaccaact ctttttccga
     4501 aggtaactgg cttcagcaga gcgcagatac caaatactgt tcttctagtg tagccgtagt
     4561 taggccacca cttcaagaac tctgtagcac cgcctacata cctcgctctg ctaatcctgt
     4621 taccagtggc tgctgccagt ggcgataagt cgtgtcttac cgggttggac tcaagacgat
     4681 agttaccgga taaggcgcag cggtcgggct gaacgggggg ttcgtgcaca cagcccagct
     4741 tggagcgaac gacctacacc gaactgagat acctacagcg tgagctatga gaaagcgcca
     4801 cgcttcccga agggagaaag gcggacaggt atccggtaag cggcagggtc ggaacaggag
     4861 agcgcacgag ggagcttcca gggggaaacg cctggtatct ttatagtcct gtcgggtttc
     4921 gccacctctg acttgagcgt cgatttttgt gatgctcgtc aggggggcgg agcctatgga
     4981 aaaacgccag caacgcggcc tttttacggt tcctggcctt ttgctggcct tttgctcaca
     5041 tgttctttcc tgcgttatcc cctgattctg tggataaccg tattaccgcc tttgagtgag
     5101 ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga gtcagtgagc gaggaagcgg
     5161 aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg gccgattcat taatgcagct
     5221 ggcacgacag gtttcccgac tggaaagcgg gcagtgagcg caacgcaatt aatgtgagtt
     5281 agctcactca ttaggcaccc caggctttac actttatgct tccggctcgt atgttgtgtg
     5341 gaattgtgag cggataacaa tttcacacag gaaacagcta tgaccatgat tacgccaagc
     5401 tatttaggtg acactataga atactcaagc tatgcatcca acgcgttggg agctctccca
     5461 tatggtcga
//
