LOCUS       sequence-489764-        4688 bp    DNA     circular SYN 20-OCT-2025
DEFINITION  In-Vitro Synthesis of mRNA encoding a Honeybee-optimized 
            mEmerald-labeled Lifeact (LA) Tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4688)
  TITLE     In-Vitro Synthesis of mRNA for Fluorescent Labeling of Honeybee 
            Embryos
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4688)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Oct 20, 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            "Unpublished"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-mEmerald(AM)
FEATURES             Location/Qualifiers
     source          1..4688
                     /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    1611..1745
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1648..1667)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1702..1721
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2500..2520)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2500..2518)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2539..2555)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2539..2555)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2552..2574)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2563..2579
                     /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(2587..2593,2594..2611,2612..2617))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2632..2653
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2770..2787)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2941..3529)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3021..3040)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3700..4491,4492..4560))
                     /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     4323..4342
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4561..4665)
                     /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 cctcgaaagg cgaagaattg ttcaccggcg tggtgccaat tttggtggaa ttggacggcg
      841 acgtgaacgg ccacaaattc tcggtgtcgg gcgaaggcga aggcgacgcc acctacggca
      901 aattgacctt gaaattcatt tgtaccaccg gcaaattgcc agtgccatgg ccaaccttgg
      961 tgaccacctt gacctacggc gtgcaatgtt tcgccagata cccagaccac atgaaacaac
     1021 acgacttctt caaatcggcc atgccagaag gctacgtgca agaaagaacc attttcttca
     1081 aagacgacgg caactacaaa accagagccg aagtgaaatt cgaaggcgac accttggtga
     1141 acaggattga attgaaaggc attgacttca aagaagatgg caacattttg ggccacaaat
     1201 tggaatacaa ctacaactcg cacaaagtgt acattaccgc cgacaaacaa aagaacggca
     1261 ttaaagtgaa cttcaaaacc agacacaaca ttgaagatgg ctcggtgcaa ttggccgacc
     1321 actaccaaca aaacacccca attggcgacg gcccagtgtt gttgccagac aaccactact
     1381 tgtcgaccca atcgaaattg tcgaaagacc caaacgaaaa gagggaccac atggtgttgt
     1441 tggaattcgt gaccgccgcc ggcattacct tgggcatgga cgaattgtac aaataacctg
     1501 cagggatcat aatcagccat accacatttg tagaggtttt acttgcttta aaaaacctcc
     1561 cacacctccc cctgaacctg aaacataaaa tgaatgcaat tgttgttgtt aacttgttta
     1621 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat
     1681 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct
     1741 ggatccccag gaagctcctc tgtgtcctca taaaccctaa cctcctctac ttgagaggac
     1801 attccaatca taggctgccc atccaccctc tgtgtcctcc tgttaattag gtcacttaac
     1861 aaaaaggaaa ttgggtaggg gtttttcaca gaccgctttc taagggtaat tttaaaatat
     1921 ctgggaagtc ccttccactg ctgtgttcca gaagtgttgg taaacagccc acaaatgtca
     1981 acagcagaaa catacaagct gtcagctttg cacaagggcc caacaccctg ctcatcaaga
     2041 agcactgtgg ttgctgtgtt agtaatgtgc aaaacaggag gcacattttc cccacctgtg
     2101 taggttccaa aatatctagt gttttcattt ttacttggat caggaaccca gcactccact
     2161 ggataagcat tatccttatc caaaacagcc ttgtggtcag tgttcatctg ctgactgtca
     2221 actgtagcat tttttggggt tacagtttga gcaggatatt tggtcctgta gtttgctaac
     2281 acaccctgca gctccaaagg ttccccacca acagcaaaaa aatgaaaatt tgacccttga
     2341 atgggttttc cagcaccatt ttcatgagtt ttttgtgtcc ctgaatgcaa gtttaacata
     2401 gcagttaccc caataacctc agttttaaca gtaacagctt cccacatcaa aatatttcca
     2461 caggttaagt cctcatttaa agagctccag cttttgttcc ctttagtgag ggttaattgc
     2521 gcgcttggcg taatcatggt catagctgtt tcctgtgtga aattgttatc cgctcacaat
     2581 tccacacaac atacgagccg gaagcataaa gtgtaaagcc tggggtgcct aatgagtgag
     2641 ctaactcaca ttaattgcgt tgcgctcact gcccgctttc cagtcgggaa acctgtcgtg
     2701 ccagctgcat taatgaatcg gccaacgcgc ggggagaggc ggtttgcgta ttgggcgctc
     2761 ttccgcttcc tcgctcactg actcgctgcg ctcggtcgtt cggctgcggc gagcggtatc
     2821 agctcactca aaggcggtaa tacggttatc cacagaatca ggggataacg caggaaagaa
     2881 catgtgagca aaaggccagc aaaaggccag gaaccgtaaa aaggccgcgt tgctggcgtt
     2941 tttccatagg ctccgccccc ctgacgagca tcacaaaaat cgacgctcaa gtcagaggtg
     3001 gcgaaacccg acaggactat aaagatacca ggcgtttccc cctggaagct ccctcgtgcg
     3061 ctctcctgtt ccgaccctgc cgcttaccgg atacctgtcc gcctttctcc cttcgggaag
     3121 cgtggcgctt tctcatagct cacgctgtag gtatctcagt tcggtgtagg tcgttcgctc
     3181 caagctgggc tgtgtgcacg aaccccccgt tcagcccgac cgctgcgcct tatccggtaa
     3241 ctatcgtctt gagtccaacc cggtaagaca cgacttatcg ccactggcag cagccactgg
     3301 taacaggatt agcagagcga ggtatgtagg cggtgctaca gagttcttga agtggtggcc
     3361 taactacggc tacactagaa gaacagtatt tggtatctgc gctctgctga agccagttac
     3421 cttcggaaaa agagttggta gctcttgatc cggcaaacaa accaccgctg gtagcggtgg
     3481 tttttttgtt tgcaagcagc agattacgcg cagaaaaaaa ggatctcaag aagatccttt
     3541 gatcttttct acggggtctg acgctcagtg gaacgaaaac tcacgttaag ggattttggt
     3601 catgagatta tcaaaaagga tcttcaccta gatcctttta aattaaaaat gaagttttaa
     3661 atcaatctaa agtatatatg agtaaacttg gtctgacagt taccaatgct taatcagtga
     3721 ggcacctatc tcagcgatct gtctatttcg ttcatccata gttgcctgac tccccgtcgt
     3781 gtagataact acgatacggg agggcttacc atctggcccc agtgctgcaa tgataccgcg
     3841 agacccacgc tcaccggctc cagatttatc agcaataaac cagccagccg gaagggccga
     3901 gcgcagaagt ggtcctgcaa ctttatccgc ctccatccag tctattaatt gttgccggga
     3961 agctagagta agtagttcgc cagttaatag tttgcgcaac gttgttgcca ttgctacagg
     4021 catcgtggtg tcacgctcgt cgtttggtat ggcttcattc agctccggtt cccaacgatc
     4081 aaggcgagtt acatgatccc ccatgttgtg caaaaaagcg gttagctcct tcggtcctcc
     4141 gatcgttgtc agaagtaagt tggccgcagt gttatcactc atggttatgg cagcactgca
     4201 taattctctt actgtcatgc catccgtaag atgcttttct gtgactggtg agtactcaac
     4261 caagtcattc tgagaatagt gtatgcggcg accgagttgc tcttgcccgg cgtcaatacg
     4321 ggataatacc gcgccacata gcagaacttt aaaagtgctc atcattggaa aacgttcttc
     4381 ggggcgaaaa ctctcaagga tcttaccgct gttgagatcc agttcgatgt aacccactcg
     4441 tgcacccaac tgatcttcag catcttttac tttcaccagc gtttctgggt gagcaaaaac
     4501 aggaaggcaa aatgccgcaa aaaagggaat aagggcgaca cggaaatgtt gaatactcat
     4561 actcttcctt tttcaatatt attgaagcat ttatcagggt tattgtctca tgagcggata
     4621 catatttgaa tgtatttaga aaaataaaca aataggggtt ccgcgcacat ttccccgaaa
     4681 agtgccac
//
