LOCUS       sequence-489770-        4679 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  In-Vitro Synthesis of mRNA encoding a Honeybee-optimized 
            mCherry-labeled Lifeact (LA) Tag.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4679)
  TITLE     In-Vitro Synthesis of mRNA for Fluorescent Labeling of Honeybee 
            Embryos
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 4679)
  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: 
            "Unpublished"
COMMENT     Sequence Label: pBSII-LA-mCherry(AM)
FEATURES             Location/Qualifiers
     source          1..4679
                     /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    1602..1736
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1639..1658)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     1693..1712
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(2491..2511)
                     /label=T3
                     /note="T3 promoter, forward primer"
     promoter        complement(2491..2509)
                     /label=T3 promoter
                     /note="promoter for bacteriophage T3 RNA polymerase"
     primer_bind     complement(2530..2546)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(2530..2546)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(2543..2565)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    2554..2570
                     /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(2578..2584,2585..2602,2603..2608))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    2623..2644
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(2761..2778)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(2932..3520)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(3012..3031)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(3691..4482,4483..4551))
                     /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     4314..4333
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     promoter        complement(4552..4656)
                     /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 cgaagaagat aacatggcca ttattaaaga attcatgaga ttcaaagtgc
      841 acatggaagg ctcggtgaac ggccacgaat tcgaaattga aggcgaaggc gaaggcagac
      901 catacgaagg cacccaaacc gccaaactga aagtgaccaa aggcggccca ctgccattcg
      961 cctgggacat tctgtcgcca caattcatgt acggctcgaa agcctacgtg aaacacccag
     1021 ccgacattcc agactacctg aaactgtcgt tcccagaagg cttcaaatgg gaaagagtga
     1081 tgaacttcga agatggcggc gtggtgaccg tgacccaaga ctcgtcgctg caagacggcg
     1141 aattcattta caaagtgaaa ctgagaggca ccaacttccc atcggacggc ccagtgatgc
     1201 aaaagaaaac catgggctgg gaagcctcgt cggaaagaat gtacccagaa gatggcgccc
     1261 tgaaaggcga aattaaacaa agactgaaac tgaaagacgg cggccactac gacgccgaag
     1321 tgaaaaccac ctacaaagcc aagaaaccag tgcaactgcc aggcgcctac aacgtgaaca
     1381 ttaaactgga cattacctcg cacaacgaag attacaccat tgtggaacaa tacgaaagag
     1441 ccgaaggcag acactcgacc ggcggcatgg acgaactgta caaataacct gcagggatca
     1501 taatcagcca taccacattt gtagaggttt tacttgcttt aaaaaacctc ccacacctcc
     1561 ccctgaacct gaaacataaa atgaatgcaa ttgttgttgt taacttgttt attgcagctt
     1621 ataatggtta caaataaagc aatagcatca caaatttcac aaataaagca tttttttcac
     1681 tgcattctag ttgtggtttg tccaaactca tcaatgtatc ttatcatgtc tggatcccca
     1741 ggaagctcct ctgtgtcctc ataaacccta acctcctcta cttgagagga cattccaatc
     1801 ataggctgcc catccaccct ctgtgtcctc ctgttaatta ggtcacttaa caaaaaggaa
     1861 attgggtagg ggtttttcac agaccgcttt ctaagggtaa ttttaaaata tctgggaagt
     1921 cccttccact gctgtgttcc agaagtgttg gtaaacagcc cacaaatgtc aacagcagaa
     1981 acatacaagc tgtcagcttt gcacaagggc ccaacaccct gctcatcaag aagcactgtg
     2041 gttgctgtgt tagtaatgtg caaaacagga ggcacatttt ccccacctgt gtaggttcca
     2101 aaatatctag tgttttcatt tttacttgga tcaggaaccc agcactccac tggataagca
     2161 ttatccttat ccaaaacagc cttgtggtca gtgttcatct gctgactgtc aactgtagca
     2221 ttttttgggg ttacagtttg agcaggatat ttggtcctgt agtttgctaa cacaccctgc
     2281 agctccaaag gttccccacc aacagcaaaa aaatgaaaat ttgacccttg aatgggtttt
     2341 ccagcaccat tttcatgagt tttttgtgtc cctgaatgca agtttaacat agcagttacc
     2401 ccaataacct cagttttaac agtaacagct tcccacatca aaatatttcc acaggttaag
     2461 tcctcattta aagagctcca gcttttgttc cctttagtga gggttaattg cgcgcttggc
     2521 gtaatcatgg tcatagctgt ttcctgtgtg aaattgttat ccgctcacaa ttccacacaa
     2581 catacgagcc ggaagcataa agtgtaaagc ctggggtgcc taatgagtga gctaactcac
     2641 attaattgcg ttgcgctcac tgcccgcttt ccagtcggga aacctgtcgt gccagctgca
     2701 ttaatgaatc ggccaacgcg cggggagagg cggtttgcgt attgggcgct cttccgcttc
     2761 ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
     2821 aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc
     2881 aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag
     2941 gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc
     3001 gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt
     3061 tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct
     3121 ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
     3181 ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actgtcgtct
     3241 tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat
     3301 tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg
     3361 ctacactaga agaacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa
     3421 aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt
     3481 ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
     3541 tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgagatt
     3601 atcaaaaagg atcttcacct agatcctttt aaattaaaaa tgaagtttta aatcaatcta
     3661 aagtatatat gagtaaactt ggtctgacag ttaccaatgc ttaatcagtg aggcacctat
     3721 ctcagcgatc tgtctatttc gttcatccat agttgcctga ctccccgtcg tgtagataac
     3781 tacgatacgg gagggcttac catctggccc cagtgctgca atgataccgc gagacccacg
     3841 ctcaccggct ccagatttat cagcaataaa ccagccagcc ggaagggccg agcgcagaag
     3901 tggtcctgca actttatccg cctccatcca gtctattaat tgttgccggg aagctagagt
     3961 aagtagttcg ccagttaata gtttgcgcaa cgttgttgcc attgctacag gcatcgtggt
     4021 gtcacgctcg tcgtttggta tggcttcatt cagctccggt tcccaacgat caaggcgagt
     4081 tacatgatcc cccatgttgt gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt
     4141 cagaagtaag ttggccgcag tgttatcact catggttatg gcagcactgc ataattctct
     4201 tactgtcatg ccatccgtaa gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
     4261 ctgagaatag tgtatgcggc gaccgagttg ctcttgcccg gcgtcaatac gggataatac
     4321 cgcgccacat agcagaactt taaaagtgct catcattgga aaacgttctt cggggcgaaa
     4381 actctcaagg atcttaccgc tgttgagatc cagttcgatg taacccactc gtgcacccaa
     4441 ctgatcttca gcatctttta ctttcaccag cgtttctggg tgagcaaaaa caggaaggca
     4501 aaatgccgca aaaaagggaa taagggcgac acggaaatgt tgaatactca tactcttcct
     4561 ttttcaatat tattgaagca tttatcaggg ttattgtctc atgagcggat acatatttga
     4621 atgtatttag aaaaataaac aaataggggt tccgcgcaca tttccccgaa aagtgccac
//
