LOCUS       sequence-491860-        4685 bp    DNA     circular SYN 04-NOV-2025
DEFINITION  In-Vitro Synthesis of mRNA encoding a Honeybee-optimized 
            mTagBFP2-labeled Lifeact (LA) Tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4685)
  TITLE     In-Vitro Synthesis of mRNA for Fluorescent Labeling of Honeybee 
            Embryos
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4685)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Nov 4, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: pBSII-LA-mTagBFP(AM)
FEATURES             Location/Qualifiers
     source          1..4685
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     rep_origin      complement(30..458)
                     /direction=LEFT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(140..161)
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     352..371
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     585..607
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     599..616
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     600..616
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     626..645
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        626..644
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     primer_bind     669..685
                     /label=pBluescriptKS
                     /note="For pBluescript vector"
     primer_bind     670..686
                     /label=KS primer
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             723..773
                     /codon_start=1
                     /product="peptide that binds filamentous actin (Riedl et 
                     al., 2008)"
                     /label=Lifeact
                     /note="first 17 amino acids of budding yeast Abp140"
                     /translation="MGVADLIKKFESISKEE"
     polyA_signal    1608..1742
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1645..1664)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1699..1718
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2497..2517)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2497..2515)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2536..2552)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2536..2552)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2549..2571)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2560..2576
                     /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(2584..2590,2591..2608,2609..2614))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2629..2650
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2767..2784)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2938..3526)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3018..3037)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3697..4488,4489..4557))
                     /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     4320..4339
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4558..4662)
                     /gene="bla"
                     /label=AmpR promoter
ORIGIN
        1 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc
       61 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga
      121 gatagggttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc
      181 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc
      241 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag
      301 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggaagaa
      361 agcgaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac
      421 cacacccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg
      481 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg
      541 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt cacgacgttg
      601 taaaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccgg
      661 gccccccctc gaggtcgacg gtatcgataa gcttgatatc gaattcctgc agcccgggca
      721 aaatgggcgt ggccgacttg attaaaaaat tcgaatcgat ttcgaaagaa gaagcggccg
      781 ccatgtcgaa aggtgaagaa ttgattaaag aaaacatgca tatgaaattg tacatggaag
      841 gcaccgtgga caaccaccac ttcaaatgca cctcggaagg cgaaggcaaa ccatacgaag
      901 gcacccaaac aatgagaatt aaagtggtgg aaggtggccc attgccattc gccttcgaca
      961 ttttggccac ctcgttcttg tacggatcga aaaccttcat taaccacacc caaggcattc
     1021 cagatttctt caaacaatcg ttcccagaag gcttcacctg ggaaagagtg acaacctacg
     1081 aagacggagg agtgttgacc gccacccaag acacctcgtt gcaagacgga tgcttgattt
     1141 acaacgtgaa aattagagga gtgaacttca catcgaatgg cccagtgatg caaaagaaaa
     1201 ccttgggttg ggaagccttc accgaaacct tgtacccagc cgatggtggt ttggaaggca
     1261 gaaacgacat ggcgttgaaa ttggtgggcg gctcgcactt gattgccaac gccaaaacca
     1321 cctacagatc gaaaaaaccg gccaaaaact tgaaaatgcc aggtgtgtac tacgtggact
     1381 acagattgga aagaattaaa gaagccaaca acgaaaccta cgtggaacaa catgaagtgg
     1441 cggtggcgag atactgcgac ttgccgtcga aattgggcca caaattgaac taacctgcag
     1501 ggatcataat cagccatacc acatttgtag aggttttact tgctttaaaa aacctcccac
     1561 acctccccct gaacctgaaa cataaaatga atgcaattgt tgttgttaac ttgtttattg
     1621 cagcttataa tggttacaaa taaagcaata gcatcacaaa tttcacaaat aaagcatttt
     1681 tttcactgca ttctagttgt ggtttgtcca aactcatcaa tgtatcttat catgtctgga
     1741 tccccaggaa gctcctctgt gtcctcataa accctaacct cctctacttg agaggacatt
     1801 ccaatcatag gctgcccatc caccctctgt gtcctcctgt taattaggtc acttaacaaa
     1861 aaggaaattg ggtaggggtt tttcacagac cgctttctaa gggtaatttt aaaatatctg
     1921 ggaagtccct tccactgctg tgttccagaa gtgttggtaa acagcccaca aatgtcaaca
     1981 gcagaaacat acaagctgtc agctttgcac aagggcccaa caccctgctc atcaagaagc
     2041 actgtggttg ctgtgttagt aatgtgcaaa acaggaggca cattttcccc acctgtgtag
     2101 gttccaaaat atctagtgtt ttcattttta cttggatcag gaacccagca ctccactgga
     2161 taagcattat ccttatccaa aacagccttg tggtcagtgt tcatctgctg actgtcaact
     2221 gtagcatttt ttggggttac agtttgagca ggatatttgg tcctgtagtt tgctaacaca
     2281 ccctgcagct ccaaaggttc cccaccaaca gcaaaaaaat gaaaatttga cccttgaatg
     2341 ggttttccag caccattttc atgagttttt tgtgtccctg aatgcaagtt taacatagca
     2401 gttaccccaa taacctcagt tttaacagta acagcttccc acatcaaaat atttccacag
     2461 gttaagtcct catttaaaga gctccagctt ttgttccctt tagtgagggt taattgcgcg
     2521 cttggcgtaa tcatggtcat agctgtttcc tgtgtgaaat tgttatccgc tcacaattcc
     2581 acacaacata cgagccggaa gcataaagtg taaagcctgg ggtgcctaat gagtgagcta
     2641 actcacatta attgcgttgc gctcactgcc cgctttccag tcgggaaacc tgtcgtgcca
     2701 gctgcattaa tgaatcggcc aacgcgcggg gagaggcggt ttgcgtattg ggcgctcttc
     2761 cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag cggtatcagc
     2821 tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag gaaagaacat
     2881 gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag gccgcgttgc tggcgttttt
     2941 ccataggctc cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg
     3001 aaacccgaca ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc
     3061 tcctgttccg accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt
     3121 ggcgctttct catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa
     3181 gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta
     3241 tcgtcttgag tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa
     3301 caggattagc agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa
     3361 ctacggctac actagaagaa cagtatttgg tatctgcgct ctgctgaagc cagttacctt
     3421 cggaaaaaga gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt
     3481 ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat
     3541 cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga ttttggtcat
     3601 gagattatca aaaaggatct tcacctagat ccttttaaat taaaaatgaa gttttaaatc
     3661 aatctaaagt atatatgagt aaacttggtc tgacagttac caatgcttaa tcagtgaggc
     3721 acctatctca gcgatctgtc tatttcgttc atccatagtt gcctgactcc ccgtcgtgta
     3781 gataactacg atacgggagg gcttaccatc tggccccagt gctgcaatga taccgcgaga
     3841 cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa gggccgagcg
     3901 cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt gccgggaagc
     3961 tagagtaagt agttcgccag ttaatagttt gcgcaacgtt gttgccattg ctacaggcat
     4021 cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc tccggttccc aacgatcaag
     4081 gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt agctccttcg gtcctccgat
     4141 cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg gttatggcag cactgcataa
     4201 ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt actcaaccaa
     4261 gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt caatacggga
     4321 taataccgcg ccacatagca gaactttaaa agtgctcatc attggaaaac gttcttcggg
     4381 gcgaaaactc tcaaggatct taccgctgtt gagatccagt tcgatgtaac ccactcgtgc
     4441 acccaactga tcttcagcat cttttacttt caccagcgtt tctgggtgag caaaaacagg
     4501 aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa tactcatact
     4561 cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga gcggatacat
     4621 atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc cccgaaaagt
     4681 gccac
//
