LOCUS       sequence-495316-        6889 bp    DNA     circular SYN 22-JAN-2026
DEFINITION  Expresses ALFA-bRho-EGFP in mammalian cells. Modified from Addgene 
            plasmid #45399.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 6889)
  AUTHORS   Hekmatara M, Thompson SL, Haggerty KN, Hagen S, Brothers BA, Daniels
            B, Luxardi G, Moshiri A, Deng WT, Robichaux MA
  TITLE     Super-resolution microscopy reveals a Rab6a-dependent trafficking 
            hub for rhodopsin at the mammalian rod photoreceptor Golgi.
  JOURNAL   bioRxiv [Preprint]. 2025 Sep 25:2025.09.24.678292. doi: 
            10.1101/2025.09.24.678292.
  PUBMED    41040232
REFERENCE   2  (bases 1 to 6889)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Jan 22, 2026 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: "bioRxiv 
            [Preprint]. 2025 Sep 25:2025.09.24.678292. doi: 
            10.1101/2025.09.24.678292."
COMMENT     Sequence Label: pcDNA3.1-N-ALFA-bRho-EGFP
FEATURES             Location/Qualifiers
     source          1..6889
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        complement(10..114)
                     /gene="bla"
                     /label=AmpR promoter
     primer_bind     complement(189..208)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     enhancer        380..759
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        760..963
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     914..934
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     primer_bind     1008..1027
                     /label=T7
                     /note="T7 promoter, forward primer"
     promoter        1008..1026
                     /label=T7 promoter
                     /note="promoter for bacteriophage T7 RNA polymerase"
     CDS             1118..1156
                     /codon_start=1
                     /product="rationally designed epitope tag that forms a 
                     stable alpha-helix (Gotzke et al., 2019)"
                     /label=ALFA-tag
                     /note="It may be beneficial to flank the ALFA-tag sequence 
                     with proline residues to preserve the helical structure."
                     /translation="SRLEEELRRRLTE"
     CDS             1169..1207
                     /codon_start=1
                     /product="segment of bovine rhodopsin containing a signal 
                     for N-linked glycosylation (Favaloro et al., 2008)"
                     /label=opsin tag
                     /note="The asparagine in the NKT tripeptide will be 
                     glycosylated if this sequence is exposed to the ER lumen."
                     /translation="GPNFYVPFSNKTG"
     CDS             join(2198..2200,2201..2203,2204..2914)
                     /codon_start=1
                     /product="enhanced GFP"
                     /label=EGFP
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTL
                     KFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDD
                     GNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK
                     VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLL
                     EFVTAAGITLGMDELYK"
     CDS             join(2198..2200,2201..2203,2204..2914)
                     /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(2243..2264)
                     /label=EGFP-N
                     /note="EGFP, reverse primer"
     primer_bind     complement(2504..2523)
                     /label=EXFP-R
                     /note="For distinguishing EGFP variants, reverse primer"
     primer_bind     2851..2872
                     /label=EGFP-C
                     /note="EGFP, forward primer"
     primer_bind     complement(2986..3003)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2992..3216
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     primer_bind     complement(3349..3368)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     protein_bind    3500..3547
                     /label=FRT
                     /bound_moiety="FLP recombinase from the Saccharomyces 
                     cerevisiae 2u plasmid"
                     /note="FLP-mediated recombination occurs in the 8-bp core 
                     sequence TCTAGAAA (Turan and Bode, 2011)."
     CDS             3555..4577
                     /codon_start=1
                     /product="hygromycin B phosphotransferase"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     CDS             3555..4577
                     /codon_start=1
                     /gene="aph(4)-Ia"
                     /product="aminoglycoside phosphotransferase from E. coli"
                     /label=HygR
                     /note="confers resistance to hygromycin"
                     /translation="KKPELTATSVEKFLIEKFDSVSDLMQLSEGEESRAFSFDVGGRGY
                     VLRVNSCADGFYKDRYVYRHFASAALPIPEVLDIGEFSESLTYCISRRAQGVTLQDLPE
                     TELPAVLQPVAEAMDAIAAADLSQTSGFGPFGPQGIGQYTTWRDFICAIADPHVYHWQT
                     VMDDTVSASVAQALDELMLWAEDCPEVRHLVHADFGSNNVLTDNGRITAVIDWSEAMFG
                     DSQYEVANIFFWRPWLACMEQQTRYFERRHPELAGSPRLRAYMLRIGLDQLYQSLVDGN
                     FDDAAWAQGRCDAIVRSGAGTVGRTQIARRSAAVWTDGCVEVLADSGNRRPSTRPRAKE
                     "
     polyA_signal    4707..4828
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(4744..4763)
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     primer_bind     4798..4817
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     complement(4877..4893)
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4877..4893)
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     primer_bind     complement(4890..4912)
                     /label=M13/pUC Reverse
                     /note="In lacZ gene"
     protein_bind    4901..4917
                     /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(4925..4931,4932..4949,4950..4955))
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4970..4991
                     /label=CAP binding site
                     /bound_moiety="E. coli catabolite activator protein"
                     /note="CAP binding activates transcription in the presence 
                     of cAMP."
     primer_bind     complement(5108..5125)
                     /label=L4440
                     /note="L4440 vector, forward primer"
     rep_origin      complement(5279..5867)
                     /direction=LEFT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     complement(5359..5378)
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     CDS             complement(join(6038..6829,6830..6889,1..9))
                     /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     6661..6680
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
ORIGIN
        1 aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat
       61 gagcggatac atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt
      121 tccccgaaaa gtgccacctg acgtcgacgg atcgggagat ctcccgatcc cctatggtgc
      181 actctcagta caatctgctc tgatgccgca tagttaagcc agtatctgct ccctgcttgt
      241 gtgttggagg tcgctgagta gtgcgcgagc aaaatttaag ctacaacaag gcaaggcttg
      301 accgacaatt gcatgaagaa tctgcttagg gttaggcgtt ttgcgctgct tcgcgatgta
      361 cgggccagat atacgcgttg acattgatta ttgactagtt attaatagta atcaattacg
      421 gggtcattag ttcatagccc atatatggag ttccgcgtta cataacttac ggtaaatggc
      481 ccgcctggct gaccgcccaa cgacccccgc ccattgacgt caataatgac gtatgttccc
      541 atagtaacgc caatagggac tttccattga cgtcaatggg tggagtattt acggtaaact
      601 gcccacttgg cagtacatca agtgtatcat atgccaagta cgccccctat tgacgtcaat
      661 gacggtaaat ggcccgcctg gcattatgcc cagtacatga ccttatggga ctttcctact
      721 tggcagtaca tctacgtatt agtcatcgct attaccatgg tgatgcggtt ttggcagtac
      781 atcaatgggc gtggatagcg gtttgactca cggggatttc caagtctcca ccccattgac
      841 gtcaatggga gtttgttttg gcaccaaaat caacgggact ttccaaaatg tcgtaacaac
      901 tccgccccat tgacgcaaat gggcggtagg cgtgtacggt gggaggtcta tataagcaga
      961 gctctctggc taactagaga acccactgct tactggctta tcgaaattaa tacgactcac
     1021 tatagggaga cccaagctgg ctagcgttta aacttaagct tggtaccgag ctcggatcca
     1081 ctagtaacgg ccgccagtgt gctggaattc caccatgagc cgtctggaag aagaactgcg
     1141 ccgccgtctg accgaaaacg gtaccgaagg cccaaacttc tacgttcctt tctccaacaa
     1201 gacgggcgtg gtgcgcagcc cgttcgaggc tccgcagtac tacctggcgg agccctggca
     1261 gttctccatg ctggccgcct acatgttcct gctgatcatg cttggcttcc cgatcaactt
     1321 cctcacgctg tacgtcacag tccagcacaa gaagcttcgc acaccgctca actacatcct
     1381 gctcaacctg gccgtggcag atctcttcat ggtcttcggt ggcttcacca ccaccctcta
     1441 cacctctctc catgggtact tcgtctttgg gccgacgggc tgcaacctcg agggcttctt
     1501 tgccaccctg ggcggtgaaa ttgcactgtg gtctctggta gtactggcga tcgagcggta
     1561 cgtggtggtg tgcaagccca tgagcaactt ccgcttcggt gagaaccacg ccatcatggg
     1621 cgtcgccttc acctgggtca tggctctggc ctgtgcggcc ccgccgctcg tcggctggtc
     1681 tagatacatc ccggagggca tgcagtgctc gtgcgggatc gattactaca cgccgcacga
     1741 ggagaccaac aatgagtcgt tcgtcatcta catgttcgtg gtccacttca tcatcccgct
     1801 gattgtcatc ttcttctgct atggccagct ggtgttcacc gtcaaggagg ctgcagccca
     1861 gcagcaggag agcgccacca ctcagaaggc cgagaaggag gtcacgcgta tggttatcat
     1921 catggtcatc gctttcctaa tctgctggct gccatatgct ggtgtggcgt tctacatctt
     1981 cacccatcag ggctctgact ttgggcccat cttcatgacc atcccggctt tctttgccaa
     2041 gacgtctgcc gtctacaacc cggtcatcta catcatgatg aacaagcagt tccggaactg
     2101 catggtcacc actctctgct gtggcaagaa cccgctgggt gacgacgagg cgtcgaccac
     2161 cgtctccaag acagagacca gccaagtggc gcctgccatg gtgagcaagg gcgaggagct
     2221 gttcaccggg gtggtgccca tcctggtcga gctggacggc gacgtaaacg gccacaagtt
     2281 cagcgtgtcc ggcgagggcg agggcgatgc cacctacggc aagctgaccc tgaagttcat
     2341 ctgcaccacc ggcaagctgc ccgtgccctg gcccaccctc gtgaccaccc tgacctacgg
     2401 cgtgcagtgc ttcagccgct accccgacca catgaagcag cacgacttct tcaagtccgc
     2461 catgcccgaa ggctacgtcc aggagcgcac catcttcttc aaggacgacg gcaactacaa
     2521 gacccgcgcc gaggtgaagt tcgagggcga caccctggtg aaccgcatcg agctgaaggg
     2581 catcgacttc aaggaggacg gcaacatcct ggggcacaag ctggagtaca actacaacag
     2641 ccacaacgtc tatatcatgg ccgacaagca gaagaacggc atcaaggtga acttcaagat
     2701 ccgccacaac atcgaggacg gcagcgtgca gctcgccgac cactaccagc agaacacccc
     2761 catcggcgac ggccccgtgc tgctgcccga caaccactac ctgagcaccc agtccgccct
     2821 gagcaaagac cccaacgaga agcgcgatca catggtcctg ctggagttcg tgaccgccgc
     2881 cgggatcact ctcggcatgg acgagctgta caaggagacc agccaagtgg cgcctgccta
     2941 agcggccgct cgagtctaga gggcccgttt aaacccgctg atcagcctcg actgtgcctt
     3001 ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc ctggaaggtg
     3061 ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt ctgagtaggt
     3121 gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat tgggaagaca
     3181 atagcaggca tgctggggat gcggtgggct ctatggcttc tgaggcggaa agaaccagct
     3241 ggggctctag ggggtatccc cacgcgccct gtagcggcgc attaagcgcg gcgggtgtgg
     3301 tggttacgcg cagcgtgacc gctacacttg ccagcgccct agcgcccgct cctttcgctt
     3361 tcttcccttc ctttctcgcc acgttcgccg gctttccccg tcaagctcta aatcgggggc
     3421 tccctttagg gttccgattt agtgctttac ggcacctcga ccccaaaaaa cttgattagg
     3481 gtgatggttc acgtacctag aagttcctat tccgaagttc ctattctcta gaaagtatag
     3541 gaacttcctt ggccaaaaag cctgaactca ccgcgacgtc tgtcgagaag tttctgatcg
     3601 aaaagttcga cagcgtctcc gacctgatgc agctctcgga gggcgaagaa tctcgtgctt
     3661 tcagcttcga tgtaggaggg cgtggatatg tcctgcgggt aaatagctgc gccgatggtt
     3721 tctacaaaga tcgttatgtt tatcggcact ttgcatcggc cgcgctcccg attccggaag
     3781 tgcttgacat tggggaattc agcgagagcc tgacctattg catctcccgc cgtgcacagg
     3841 gtgtcacgtt gcaagacctg cctgaaaccg aactgcccgc tgttctgcag ccggtcgcgg
     3901 aggccatgga tgcgatcgct gcggccgatc ttagccagac gagcgggttc ggcccattcg
     3961 gaccgcaagg aatcggtcaa tacactacat ggcgtgattt catatgcgcg attgctgatc
     4021 cccatgtgta tcactggcaa actgtgatgg acgacaccgt cagtgcgtcc gtcgcgcagg
     4081 ctctcgatga gctgatgctt tgggccgagg actgccccga agtccggcac ctcgtgcacg
     4141 cggatttcgg ctccaacaat gtcctgacgg acaatggccg cataacagcg gtcattgact
     4201 ggagcgaggc gatgttcggg gattcccaat acgaggtcgc caacatcttc ttctggaggc
     4261 cgtggttggc ttgtatggag cagcagacgc gctacttcga gcggaggcat ccggagcttg
     4321 caggatcgcc gcggctccgg gcgtatatgc tccgcattgg tcttgaccaa ctctatcaga
     4381 gcttggttga cggcaatttc gatgatgcag cttgggcgca gggtcgatgc gacgcaatcg
     4441 tccgatccgg agccgggact gtcgggcgta cacaaatcgc ccgcagaagc gcggccgtct
     4501 ggaccgatgg ctgtgtagaa gtactcgccg atagtggaaa ccgacgcccc agcactcgtc
     4561 cgagggcaaa ggaatagcac gtactacgag atttcgattc caccgccgcc ttctatgaaa
     4621 ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag cgcggggatc
     4681 tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat ggttacaaat
     4741 aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat tctagttgtg
     4801 gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc tctagctaga
     4861 gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg ctcacaattc
     4921 cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa tgagtgagct
     4981 aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac ctgtcgtgcc
     5041 agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt gggcgctctt
     5101 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag
     5161 ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca
     5221 tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt
     5281 tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc
     5341 gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct
     5401 ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
     5461 tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca
     5521 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact
     5581 atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta
     5641 acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta
     5701 actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag ccagttacct
     5761 tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
     5821 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga
     5881 tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca
     5941 tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga agttttaaat
     6001 caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta atcagtgagg
     6061 cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt
     6121 agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
     6181 acccacgctc accggctcca gatttatcag caataaacca gccagccgga agggccgagc
     6241 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt tgccgggaag
     6301 ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt gctacaggca
     6361 tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc caacgatcaa
     6421 ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga
     6481 tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
     6541 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag tactcaacca
     6601 agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg tcaatacggg
     6661 ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa cgttcttcgg
     6721 ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg
     6781 cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag
     6841 gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttg
//
