LOCUS       sequence-329482-        6920 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 6920)
  AUTHORS   Le Y, Miller JL, Sauer B
  TITLE     GFPcre fusion vectors with enhanced expression.
  JOURNAL   Anal Biochem. 1999 Jun 1. 270(2):334-6.
  PUBMED    10334853
REFERENCE   2  (bases 1 to 6920)
  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: "Anal 
            Biochem. 1999 Jun 1. 270(2):334-6."
COMMENT     Sequence Label: pBS594 promoterless EGFPcre
FEATURES             Location/Qualifiers
     source          1..6920
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     53..71
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     primer_bind     complement(109..131)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     231..250
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     444..466
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     458..475
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     459..475
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     regulatory      509..518
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             join(515..517,518..520,521..1231)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(515..517,518..520,521..1231)
                     /codon_start=1
                     /product="the original enhanced GFP (Yang et al., 1996)"
                     /label=EGFP
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     primer_bind     complement(821..840)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     1168..1189
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     CDS             1286..2317
                     /codon_start=1
                     /product="site-specific recombinase"
                     /label=Cre
                     /note="Cre is a site-specific recombinase from 
                     bacteriophage P1. Recombination occurs at loxP sequences."
                     /translation="MANLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKML
                     LSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGL
                     PRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAF
                     LGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVE
                     RWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQ
                     RYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLE
                     DGD"
     RBS             3969..3977
                     /label=Shine-Dalgarno sequence
                     /note="full consensus sequence for ribosome-binding sites 
                     upstream of start codons in E. coli; complementary to a 
                     region in the 3' end of the 16S rRNA (Chen et al., 1994)"
     primer_bind     complement(4779..4795)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4779..4795)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4792..4814)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4803..4819
                     /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(join(4827..4833,4834..4851,4852..4857))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4872..4893
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5010..5027)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5181..5769)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5261..5280)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(5940..6731,6732..6800))
                     /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     6563..6582
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(6801..6905)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 agtgccacct gacgtctaag aaaccattat tatcatgaca ttaacctata aaaataggcg
       61 tatcacgagg ccctttcgtc tcgcgcgttt cggtgatgac ggtgaaaacc tctgacacat
      121 gcagctcccg gagacggtca cagcttgtct gtaagcggat gccgggagca gacaagcccg
      181 tcagggcgcg tcagcgggtg ttggcgggtg tcggggctgg cttaactatg cggcatcaga
      241 gcagattgta ctgagagtgc accatatgcg gtgtgaaata ccgcacagat gcgtaaggag
      301 aaaataccgc atcaggcgcc attcgccatt caggctgcgc aactgttggg aagggcgatc
      361 ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt
      421 aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgccaa
      481 gcttctcgag tctagatctg caccggtcgc caccatggtg agcaagggcg aggagctgtt
      541 caccggggtg gtgcccatcc tggtcgagct ggacggcgac gtaaacggcc acaagttcag
      601 cgtgtccggc gagggcgagg gcgatgccac ctacggcaag ctgaccctga agttcatctg
      661 caccaccggc aagctgcccg tgccctggcc caccctcgtg accaccctga cctacggcgt
      721 gcagtgcttc agccgctacc ccgaccacat gaagcagcac gacttcttca agtccgccat
      781 gcccgaaggc tacgtccagg agcgcaccat cttcttcaag gacgacggca actacaagac
      841 ccgcgccgag gtgaagttcg agggcgacac cctggtgaac cgcatcgagc tgaagggcat
      901 cgacttcaag gaggacggca acatcctggg gcacaagctg gagtacaact acaacagcca
      961 caacgtctat atcatggccg acaagcagaa gaacggcatc aaggtgaact tcaagatccg
     1021 ccacaacatc gaggacggca gcgtgcagct cgccgaccac taccagcaga acacccccat
     1081 cggcgacggc cccgtgctgc tgcccgacaa ccactacctg agcacccagt ccgccctgag
     1141 caaagacccc aacgagaagc gcgatcacat ggtcctgctg gagttcgtga ccgccgccgg
     1201 gatcactctc ggcatggacg agctgtacaa gtccggtaca gctctcgacg gagaaagctc
     1261 aggctctggc tcagagtctg actccatggc caatttactg accgtacacc aaaatttgcc
     1321 tgcattaccg gtcgatgcaa cgagtgatga ggttcgcaag aacctgatgg acatgttcag
     1381 ggatcgccag gcgttttctg agcatacctg gaaaatgctt ctgtccgttt gccggtcgtg
     1441 ggcggcatgg tgcaagttga ataaccggaa atggtttccc gcagaacctg aagatgttcg
     1501 cgattatctt ctatatcttc aggcgcgcgg tctggcagta aaaactatcc agcaacattt
     1561 gggccagcta aacatgcttc atcgtcggtc cgggctgcca cgaccaagtg acagcaatgc
     1621 tgtttcactg gttatgcggc ggatccgaaa agaaaacgtt gatgccggtg aacgtgcaaa
     1681 acaggctcta gcgttcgaac gcactgattt cgaccaggtt cgttcactca tggaaaatag
     1741 cgatcgctgc caggatatac gtaatctggc atttctgggg attgcttata acaccctgtt
     1801 acgtatagcc gaaattgcca ggatcagggt taaagatatc tcacgtactg acggtgggag
     1861 aatgttaatc catattggca gaacgaaaac gctggttagc accgcaggtg tagagaaggc
     1921 acttagcctg ggggtaacta aactggtcga gcgatggatt tccgtctctg gtgtagctga
     1981 tgatccgaat aactacctgt tttgccgggt cagaaaaaat ggtgttgccg cgccatctgc
     2041 caccagccag ctatcaactc gcgccctgga agggattttt gaagcaactc atcgattgat
     2101 ttacggcgct aaggatgact ctggtcagag atacctggcc tggtctggac acagtgcccg
     2161 tgtcggagcc gcgcgagata tggcccgcgc tggagtttca ataccggaga tcatgcaagc
     2221 tggtggctgg accaatgtaa atattgtcat gaactatatc cgtaacctgg atagtgaaac
     2281 aggggcaatg gtgcgcctgc tggaagatgg cgattagcca ttaacgcgta aatgattgct
     2341 ataattattt gatatttatg gtgacatatg agaaaggatt tcaacatcga cggaaaatat
     2401 gtagtgctgt ctgtaagcac taatattcag tcgccagccg tcattgtcac tgtaaagctg
     2461 agcgatagaa tgcctgatat tgactcaata tccgttgcgt ttcctgtcaa aagtatgcgt
     2521 agtgctgaac atttcgtgat gaatgccacc gaggaagaag cacggcgcgg ttttgctaaa
     2581 gtgatgtctg agtttggcga actcttgggt aaggttggaa ttgtcgaggg atctcggaat
     2641 ggaccccaac tgctcctgct ccaccggtaa gactcccgat ccttggtctt tagaatacca
     2701 agttgggacc gcagagcgga atccccgagt tgtagaggct tggcgggaat aggcaccttt
     2761 agttggcgat tcattccggt tctttctaga atccgctctt gcaaaagcct tcattagtta
     2821 cgagtattgt cgaacgggtc ctttggcggg gttggggcta ggatttagac gcgcaaatgt
     2881 ccggttcctg atcacccagt tagtggggac atctgggttg agtcccaggc attactaaac
     2941 ttactgtgaa ttgcttgaat taagaaagag gtgaaggacc tttatgtctt gggactcaaa
     3001 gacataatcc ctgacttaac ctgtgaggag aaaagtgggg ctaggctccc tgcagctccg
     3061 aggaggactt agtgaactga gccgggactc gtggtgttgg ccactgctgt aatgctgcct
     3121 ccctcatgct gtcttctttc tcctccccag gcggctcctg cacttgcacc agctcctgcg
     3181 cctgcaagaa ctgcaagtgc acctcctgca agaagagtga gttgggacac cttgggtggc
     3241 ggctaaggct aggggcgggg aactcctaca aaactggctc tgagaaatgt cctttgcttc
     3301 ccggaggcca ttgtattgtc tcggggacag aactatacag agaactattt aaaaaaaccg
     3361 aggtcttctc tgttggggac aggaagcaga ggtcttcagc caggctgacc tcttcctcct
     3421 cctttctagg ctgctgctcc tgctgtcccg tgggctgctc caaatgtgcc cagggctgtg
     3481 tctgcaaagg cgccgcggac aagtgcacgt gctgtgcctg atgtgacgaa cagcgctgcc
     3541 accacgtgta aatagtatcg gaccaaccca gcgtcttcct atacagttcc accctgttta
     3601 ctaaaccccc gttttctacc gagtacgtga ataataaaag cctgtttgag tctaactctg
     3661 gttttcttgg tgtggtttgg caataagaaa ctggggtgac ttgatagtct ggggatctgg
     3721 ttttggaccc cctcgtgcct ttacctcccg cctctggccc tcacagaggg gtaatgtctt
     3781 tgggtaaagc caagctatcc cataagcttc ctcatggaaa acagctgtgt gcctggtcag
     3841 gtcttgtgtt aggggaaggg agggagggag ggtggtcctg gaagacttga tgtcccatcc
     3901 tggaagcaca gaaaatctta tcatttatat tctctttgga ttctggtttc ctctcacatt
     3961 gttgggtata aggaggtggg tatgaaaatt aacatagggt gtctcgtatg ctaggcaggg
     4021 actaacactg aactagattc cagaggggct ttcttggctg ttttgacaca agttgcttac
     4081 cttgtcctag aactcattct gcagtccagt ctgaccttga gtttttgatt ctcctgcctc
     4141 agcttcccaa gtacctggga ttacaggagt gactgtgtca ccaggccatc tcctattcag
     4201 ccaccaaatg cagacactat tgcatatgcc atcaagattt tgctgaaagg accctgataa
     4261 agctgtctcc tgtgaggctt tgccagtgcc cagcaaacac agaagtggat gctcacagtc
     4321 agctattgga tggaacacag ggcccccaat ggaggagcta gagaaagtac ccaaggagct
     4381 aaagggatct gcaaccctat aggtggaaca gcaatctgaa ctaaccagta ccccctgaac
     4441 tcgtgtatct agcttcgtat gtagcagaag atggcctagt tggccatcac tgggaagaga
     4501 ggacccttgg tctagcaaac tatatgcccc agttcagggg aaagccaggg ccaagaagtg
     4561 ggagtgagtg ggtaggggag cggggtgggg gtaggggtgg ggtatagggg acttttggga
     4621 tagcatttga aatataaacg aagaaaatat ccaaaaaaaa aaagatggaa aaaaaagtga
     4681 taaagagcgt gggtcctgcc tgtaatcaag atatttgaga tgtagcagca ggaaggccag
     4741 gaattggtcg agggtaccga gctcgaattc gtaatcatgt catagctgtt tcctgtgtga
     4801 aattgttatc cgctcacaat tccacacaac atacgagccg gaagcataaa gtgtaaagcc
     4861 tggggtgcct aatgagtgag ctaactcaca ttaattgcgt tgcgctcact gcccgctttc
     4921 cagtcgggaa acctgtcgtg ccagctgcat taatgaatcg gccaacgcgc ggggagaggc
     4981 ggtttgcgta ttgggcgctc ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt
     5041 cggctgcggc gagcggtatc agctcactca aaggcggtaa tacggttatc cacagaatca
     5101 ggggataacg caggaaagaa catgtgagca aaaggccagc aaaaggccag gaaccgtaaa
     5161 aaggccgcgt tgctggcgtt tttccatagg ctccgccccc ctgacgagca tcacaaaaat
     5221 cgacgctcaa gtcagaggtg gcgaaacccg acaggactat aaagatacca ggcgtttccc
     5281 cctggaagct ccctcgtgcg ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc
     5341 gcctttctcc cttcgggaag cgtggcgctt tctcatagct cacgctgtag gtatctcagt
     5401 tcggtgtagg tcgttcgctc caagctgggc tgtgtgcacg aaccccccgt tcagcccgac
     5461 cgctgcgcct tatccggtaa ctatcgtctt gagtccaacc cggtaagaca cgacttatcg
     5521 ccactggcag cagccactgg taacaggatt agcagagcga ggtatgtagg cggtgctaca
     5581 gagttcttga agtggtggcc taactacggc tacactagaa gaacagtatt tggtatctgc
     5641 gctctgctga agccagttac cttcggaaaa agagttggta gctcttgatc cggcaaacaa
     5701 accaccgctg gtagcggtgg tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa
     5761 ggatctcaag aagatccttt gatcttttct acggggtctg acgctcagtg gaacgaaaac
     5821 tcacgttaag ggattttggt catgagatta tcaaaaagga tcttcaccta gatcctttta
     5881 aattaaaaat gaagttttaa atcaatctaa agtatatatg agtaaacttg gtctgacagt
     5941 taccaatgct taatcagtga ggcacctatc tcagcgatct gtctatttcg ttcatccata
     6001 gttgcctgac tccccgtcgt gtagataact acgatacggg agggcttacc atctggcccc
     6061 agtgctgcaa tgataccgcg agacccacgc tcaccggctc cagatttatc agcaataaac
     6121 cagccagccg gaagggccga gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag
     6181 tctattaatt gttgccggga agctagagta agtagttcgc cagttaatag tttgcgcaac
     6241 gttgttgcca ttgctacagg catcgtggtg tcacgctcgt cgtttggtat ggcttcattc
     6301 agctccggtt cccaacgatc aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg
     6361 gttagctcct tcggtcctcc gatcgttgtc agaagtaagt tggccgcagt gttatcactc
     6421 atggttatgg cagcactgca taattctctt actgtcatgc catccgtaag atgcttttct
     6481 gtgactggtg agtactcaac caagtcattc tgagaatagt gtatgcggcg accgagttgc
     6541 tcttgcccgg cgtcaatacg ggataatacc gcgccacata gcagaacttt aaaagtgctc
     6601 atcattggaa aacgttcttc ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc
     6661 agttcgatgt aacccactcg tgcacccaac tgatcttcag catcttttac tttcaccagc
     6721 gtttctgggt gagcaaaaac aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca
     6781 cggaaatgtt gaatactcat actcttcctt tttcaatatt attgaagcat ttatcagggt
     6841 tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
     6901 ccgcgcacat ttccccgaaa
//
