LOCUS       sequence-324721-        6486 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  To generate a stable acceptor cell line with an improved BxbI 
            attP-GA site at the AASV1 locus, between an exogenous human pRPL30 
            promoter and a tripartite V5/SNAPtag/Blast reporter..
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6486)
  TITLE     Huang Lab Starport System Plasmids
REFERENCE   2  (bases 1 to 6486)
  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"
COMMENT     Sequence Label: StarPort02
FEATURES             Location/Qualifiers
     source          1..6486
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     misc_feature    587..1088
                     /label=HA-L
                     /note="left homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     protein_bind    1151..1184
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     protein_bind    1802..1835
                     /label=loxP
                     /bound_moiety="Cre recombinase"
                     /note="Cre-mediated recombination occurs in the 8-bp core 
                     sequence (ATGTATGC) (Shaw et al., 2021)."
     protein_bind    2035..2082
                     /label=Bxb1 attP
                     /bound_moiety="Mycobacteriophage Bxb1 integrase"
                     /note="Minimal attP site for the Mycopbacteriophage Bxb1 
                     integrase (Voutev and Mann, 2017)"
     CDS             2254..2295
                     /codon_start=1
                     /product="epitope tag from simian virus 5"
                     /label=V5 tag
                     /translation="GKPIPNPLLGLDST"
     CDS             2296..2838
                     /codon_start=1
                     /product="human O6-alkylguanine-DNA-alkyltransferase 
                     variant that forms covalent bonds with benzylguanine 
                     derivatives"
                     /label=SNAP-tag(R)
                     /note="fast-reacting variant, optimized for mammalian 
                     expression"
                     /translation="DKDCEMKRTTLDSPLGKLELSGCEQGLHRIIFLGKGTSAADAVEV
                     PAPAAVLGGPEPLMQATAWLNAYFHQPEAIEEFPVPALHHPVFQQESFTRQVLWKLLKV
                     VKFGEVISYSHLAALAGNPAATAAVKTALSGNPVPILIPCHRVVQGDLDVGGYEGGLAV
                     KEWLLAHEGHRLGKPGLG"
     CDS             3010..3063
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             3070..3465
                     /codon_start=1
                     /gene="Aspergillus terreus BSD"
                     /product="blasticidin S deaminase"
                     /label=BSD
                     /note="confers resistance to blasticidin"
                     /translation="MAKPLSQEESTLIERATATINSIPISEDYSVASAALSSDGRIFTG
                     VNVYHFTGGPCAELVVLGTAAAAAAGNLTCIVAIGNENRGILSPCGRCRQVLLDLHPGI
                     KAIVKDSDGQPTAVGIRELLPSGYVWEG"
     polyA_signal    3476..3610
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(3500..3519)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     3554..3573
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     protein_bind    3908..3941
                     /label=FRT (minimal)
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="supports FLP-mediated excision but not integration 
                     (Turan and Bode, 2011)"
     misc_feature    4113..4642
                     /label=HA-R
                     /note="right homology arm from the adeno-associated virus 
                     integration site (AAVS1) within intron 1 of the human 
                     PPP1R12C gene"
     CDS             complement(4939..5754)
                     /codon_start=1
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     CDS             complement(4939..5754)
                     /codon_start=1
                     /gene="aph(3')-Ia"
                     /product="aminoglycoside phosphotransferase"
                     /label=KanR
                     /note="confers resistance to kanamycin in bacteria or G418 
                     (Geneticin(R)) in eukaryotes"
                     /translation="MSHIQRETSCSRPRLNSNMDADLYGYKWARDNVGQSGATIYRLYG
                     KPDAPELFLKHGKGSVANDVTDEMVRLNWLTEFMPLPTIKHFIRTPDDAWLLTTAIPGK
                     TAFQVLEEYPDSGENIVDALAVFLRRLHSIPVCNCPFNSDRVFRLAQAQSRMNNGLVDA
                     SDFDDERNGWPVEQVWKEMHKLLPFSPDSVVTHGDFSLDNLIFDEGKLIGCIDVGRVGI
                     ADRYQDLAILWNCLGEFSPSLQKRLFQKYGIDNPDMNKLQFHLMLDEFF"
     primer_bind     5662..5681
                     /label=Kan-R
                     /note="Kanamycin resistance gene, reverse primer"
     primer_bind     5746..5765
                     /label=pENTR-R
                     /note="pENTR vectors, reverse primer"
     rep_origin      join(5979..6486,1..81)
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     join(6468..6486,1)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
ORIGIN
        1 tatagtcctg tcgggtttcg ccacctctga cttgagcgtc gatttttgtg atgctcgtca
       61 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt
      121 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt
      181 gcggccgcca atccacaacc agtaagggct ggcccaggtg tctggcccca cagctgagct
      241 cctaaccgca ccgagcctct ccaaaacatg gtcttttttt gctccccact gccaccacca
      301 tcaccgtccc ctgcctcatc cagtccccct tcccattctc cagagacttc ctgggccatc
      361 tccatgacct tccagaccag cctgggcttc actctcaaat ccaatgcttt ctctgacctg
      421 cattctctcc cctgggcctg tgtcgctttc tgtctgcagc ttgtggcctg ggtcacctct
      481 acggctggcc cagatccttc cctgccgcct ccttcaggtt ccgtcttcct ccactccctc
      541 ttccccttgc tctctgctgt gttgctgccc aaggatgctc tttccggagc acttccttct
      601 cggcgctgca ccacgtgatg tcctctgagc ggatcctccc cgtgtctggg tcctctccgg
      661 gcatctctcc tccctcaccc aaccccatgc cgtcttcact cgctgggttc ccttttcctt
      721 ctccttctgg ggcctgtgcc atctctcgtt tcttaggatg gccttctccg acggatgtct
      781 cccttgcgtc ccgcctcccc ttcttgtagg cctgcatcat caccgttttt ctggacaacc
      841 ccaaagtacc ccgtctgcct ggctttagcc acctctccat cctcttgctt tctttgcctg
      901 gacaccccgt tctcctgtgg attcgggtca cctctcactc ctttcatttg ggcagctccc
      961 ctacccccct tacctctcta gtctgtgcta gctcttccag ccccctgtca tggcatcttc
     1021 caggggtccg agagctcagc tagtcttctt cctccaaccc gggcccctat gtccacttca
     1081 ggacagcatg tttgctgcct ccagggatcc tgtgtccccg agctgggacc accttatatt
     1141 cccaggccct gaagttccta ttctctagaa agtataggaa cttcctgaac tggccgataa
     1201 ttgcagacga tcgaattcga gttggcgcgc ctgtcattct aaatctctct ttcagcctaa
     1261 agctttttcc ccgtatcccc ccaggtgtct gcaggctcaa agagcagcga gaagcgttca
     1321 gaggaaagcg atcccgtgcc accttccccg tgcccgggct gtccccgcac gctgccggct
     1381 cggggatgcg gggggagcgc cggaccggag cggagccccg ggcggctcgc tgctgccccc
     1441 tagcggggga gggacgtaat tacatccctg ggggctttgg gagggggctg tccccgtgag
     1501 ctcccaggcg cgcctgtcat tctaaatctc tctttcagcc taaagctttt tccccgtatc
     1561 cccccaggtg tctgcaggct caaagagcag cgagaagcgt tcagaggaaa gcgatcccgt
     1621 gccaccttcc ccgtgcccgg gctgtccccg cacgctgccg gctcggggat gcggggggag
     1681 cgccggaccg gagcggagcc ccgggcggct cgctgctgcc ccctagcggg ggagggacgt
     1741 aattacatcc ctgggggctt tggggggggg ctgtccccgt gagctcaata aaagagccca
     1801 cataacttcg tataatgtat gctatacgaa gttataacgc cccgcagcca ttctagctag
     1861 cggtaccaat agcaaccggc agctgccctc cgcttttgct ccgccccttc tgcttgcgat
     1921 ctgtttccgc ttccggtccc gcagttccgg ctctgccgtg aagagctttg cattgtggga
     1981 agtctttcct ttctcgttcc ccggccatct tagcggctgc tgttggtgag tggtggtttg
     2041 tctggtcaac caccgcggac tcagtggtgt acggtacaaa ccaggagctt tgcgggttgc
     2101 catttgtaac tccggatcct aaaattcctg tcctgttctc tgtctcttct aggttggggg
     2161 ccgtcccgct cctaaggcag gaatatgatg gagactgaca ccctcttgct ctgggtcctg
     2221 ctgctgtggg ttccgggtag taccggtgac atgggtaagc ctatccctaa ccctctcctc
     2281 ggtctggatt ctacggacaa agactgcgaa atgaagcgca ccaccctgga tagccctctg
     2341 ggcaagctgg aactgtctgg gtgcgaacag ggcctgcacc gtatcatctt cctgggcaaa
     2401 ggaacatctg ccgccgacgc cgtggaagtg cctgccccag ccgccgtgct gggcggacca
     2461 gagccactga tgcaggccac cgcctggctc aacgcctact ttcaccagcc tgaggccatc
     2521 gaggagttcc ctgtgccagc cctgcaccac ccagtgttcc agcaggagag ctttacccgc
     2581 caggtgctgt ggaaactgct gaaagtggtg aagttcggag aggtcatcag ctacagccac
     2641 ctggccgccc tggccggcaa tcccgccgcc accgccgccg tgaaaaccgc cctgagcgga
     2701 aatcccgtgc ccattctgat cccctgccac cgggtggtgc agggcgacct ggacgtgggg
     2761 ggctacgagg gcgggctcgc cgtgaaagag tggctgctgg cccacgaggg ccacagactg
     2821 ggcaagcctg ggctgggtgg ttctaacgcc gtgggccagg atacgcagga ggtgatcgtc
     2881 gtgccccaca gcttgccctt caaggtcgtg gttattagtg ctatcctggc gttggtggtg
     2941 ctgactatta tctcccttat catacttatc atgctgtggc aaaaaaaacc ccgaggatcc
     3001 ggaagcggag agggcagagg aagtctgcta acatgcggtg acgtcgagga gaatcctgga
     3061 cctggatcca tggccaagcc tttgtctcaa gaagaatcca ccctcattga aagagcaacg
     3121 gctacaatca acagcatccc catctctgaa gactacagcg tcgccagcgc agctctctct
     3181 agcgacggcc gcatcttcac tggtgtcaat gtatatcatt ttactggggg accttgtgca
     3241 gaactcgtgg tgctgggcac tgctgctgct gcggcagctg gcaacctgac ttgtatcgtc
     3301 gcgatcggaa atgagaacag gggcatcttg agcccctgcg gacggtgccg acaggtgctt
     3361 ctcgatctgc atcctgggat caaagccata gtgaaggaca gtgatggaca gccgacggca
     3421 gttgggattc gtgaattgct gccctctggt tatgtgtggg agggctggat cctaagatcc
     3481 agacatgata agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
     3541 atgctttatt tgtgaaattt gtgatgctat tgctttattt gtaaccatta taagctgcaa
     3601 taaacaagtt aacaacaaca attgcattca ttttatgttt caggttcagg gggaggtgtg
     3661 ggaggttttt taaagcaagt aaaacctcta caaatgtggt atggctgatt atgatcgctg
     3721 ctgaacaggt tgactattaa ttgtgtctgc ttgatgtgga caccaggtgg cgctggacat
     3781 cagatttgga gaggcagttg tctagggaac cgggctctgt gccagcgcag gaggcaggct
     3841 ggctctccta ttccagggat gctcatccag gaaggaaagg ttgcatgctg gacacactaa
     3901 ccttacagaa gttcctattc tctagaaagt ataggaactt cgagtgattg gtgacagaaa
     3961 agccccatcc ttaggcctcc tccttcctag tctcctgata ttgggtctaa cccccacctc
     4021 ctgttaggca gattccttat ctggtgacac acccccattt cctggagcca tctctctcct
     4081 tgccagaacc tctaaggttt gcttacgatg gagccagaga ggatcctggg agggagagct
     4141 tggcaggggg tgggagggaa gggggggatg cgtgacctgc ccggttctca gtggccaccc
     4201 tgcgctaccc tctcccagaa cctgagctgc tctgacgcgg ccgtctggtg cgtttcactg
     4261 atcctggtgc tgcagcttcc ttacacttcc caagaggaga agcagtttgg aaaaacaaaa
     4321 tcagaataag ttggtcctga gttctaactt tggctcttca cctttctagt ccccaattta
     4381 tattgttcct ccgtgcgtca gttttacctg tgagataagg ccagtagcca gccccgtcct
     4441 ggcagggctg tggtgaggag gggggtgtcc gtgtggaaaa ctccctttgt gagaatggtg
     4501 cgtcctaggt gttcaccagg tcgtggccgc ctctactccc tttctctttc tccatccttc
     4561 tttccttaaa gagtccccag tgctatctgg gacatattcc tccgcccaga gcagggtccc
     4621 gcttccctaa ggccctgctc tgggcttctg ggtttgagtc cttggcaagc ccaggagagg
     4681 cgctcaggct tccctgtccc ccttcctcgt ccaccatctc atgcccctgg ctctcctgcc
     4741 ccttccctac aggggttcct ggctctgctc tacctactgc attaaaaaaa aatttttttt
     4801 tttctaagac aaggtctgac tctgtctccc aggctggagt gcagtggtgc aatcacgctc
     4861 atagcctcaa cctcccgggc tccagtgggc ctcccacctc cgagcggccg cgtgttacaa
     4921 ccaattaacc aattctgatt agaaaaactc atcgagcatc aaatgaaact gcaatttatt
     4981 catatcagga ttatcaatac catatttttg aaaaagccgt ttctgtaatg aaggagaaaa
     5041 ctcaccgagg cagttccata ggatggcaag atcctggtat cggtctgcga ttccgactcg
     5101 tccaacatca atacaaccta ttaatttccc ctcgtcaaaa ataaggttat caagtgagaa
     5161 atcaccatga gtgacgactg aatccggtga gaatggcaaa agcttatgca tttctttcca
     5221 gacttgttca acaggccagc cattacgctc gtcatcaaaa tcactcgcat caaccaaacc
     5281 gttattcatt cgtgattgcg cctgagcgag gcgaaatacg cgatcgctgt taaaaggaca
     5341 attacaaaca ggaatcgaat gcaaccggcg caggaacact gccagcgcat caacaatatt
     5401 ttcacctgaa tcaggatatt cttctaatac ctggaatgct gttttcccgg ggatcgcagt
     5461 ggtgagtaac catgcatcat caggagtacg gataaaatgc ttgatggtcg gaagaggcat
     5521 aaattccgtc agccagttta gtctgaccat ctcatctgta acatcattgg caacgctacc
     5581 tttgccatgt ttcagaaaca actctggcgc atcgggcttc ccatacaatc gatagattgt
     5641 cgcacctgat tgcccgacat tatcgcgagc ccatttatac ccatataaat cagcatccat
     5701 gttggaattt aatcgcggcc tggagcaaga cgtttcccgt tgaatatggc tcataacacc
     5761 ccttgtatta ctgtttatgt aagcagacag ttttattgtt catgatgata tatttttatc
     5821 ttgtgcaatg taacatcaga gattttgaga cacaacgtgg ctttgttgaa taaatcgaac
     5881 ttttgctgag ttgaaggatc agtcatgacc aaaatccctt aacgtgagtt ttcgttccac
     5941 tgagcgtcag accccgtaga aaagatcaaa ggatcttctt gagatccttt ttttctgcgc
     6001 gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag cggtggtttg tttgccggat
     6061 caagagctac caactctttt tccgaaggta actggcttca gcagagcgca gataccaaat
     6121 actgttcttc tagtgtagcc gtagttaggc caccacttca agaactctgt agcaccgcct
     6181 acatacctcg ctctgctaat cctgttacca gtggctgctg ccagtggcga taagtcgtgt
     6241 cttaccgggt tggactcaag acgatagtta ccggataagg cgcagcggtc gggctgaacg
     6301 ggggggtcgt gcacacagcc cagcttggag cgaacgacct acaccgaact gagataccta
     6361 cagcgtgagc tatgagaaag cgccacgctt cccgaaggga gaaaggcgga caggtatccg
     6421 gtaagcggca gggtcggaac aggagagcgc acgagggagc ttccaggggg aaacgcctgg
     6481 tatctt
//
