LOCUS       sequence-472495-        5856 bp    DNA     circular SYN 13-MAY-2025
DEFINITION  AAV Transfection backbone for Nluc-HaloTag(R) protein.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5856)
  TITLE     Promega Early Access Collection
REFERENCE   2  (bases 1 to 5856)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported May 13, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: pNL.AAV [Nluc-HaloTag/CMV] Vector
FEATURES             Location/Qualifiers
     source          1..5856
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     repeat_region   1..130
                     /label=AAV2 ITR
     protein_bind    137..157
                     /gene="mutant version of attB"
                     /label=attB4
                     /bound_moiety="BP Clonase(TM)"
                     /note="core recombination site for the Gateway(R) BP 
                     reaction"
     enhancer        218..521
                     /label=CMV enhancer
                     /note="human cytomegalovirus immediate early enhancer"
     promoter        522..725
                     /label=CMV promoter
                     /note="human cytomegalovirus (CMV) immediate early 
                     promoter"
     primer_bind     676..696
                     /label=CMV-F
                     /note="Human CMV immediate early promoter, forward primer"
     protein_bind    746..770
                     /gene="mutant version of attB"
                     /label=attB1
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     regulatory      771..780
                     /label=Kozak sequence
                     /note="vertebrate consensus sequence for strong initiation 
                     of translation (Kozak, 1987)"
                     /regulatory_class="other"
     CDS             777..1289
                     /codon_start=1
                     /product="NanoLuc(R) luciferase"
                     /label=Nluc
                     /note="human codon-optimized"
                     /translation="MVFTLEDFVGDWRQTAGYNLDQVLEQGGVSSLFQNLGVSVTPIQR
                     IVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIFKVVYPVDDHHFKVILHYGTLVIDGV
                     TPNMIDYFGRPYEGIAVFDGKKITVTGTLWNGNKIIDERLINPDGSLLFRVTINGVTGW
                     RLCERILA"
     CDS             1338..2228
                     /codon_start=1
                     /product="modified bacterial dehalogenase that forms 
                     covalent bonds with chloroalkane derivatives"
                     /label=HaloTag(R)
                     /translation="MAEIGTGFPFDPHYVEVLGERMHYVDVGPRDGTPVLFLHGNPTSS
                     YVWRNIIPHVAPTHRCIAPDLIGMGKSDKPDLGYFFDDHVRFMDAFIEALGLEEVVLVI
                     HDWGSALGFHWAKRNPERVKGIAFMEFIRPIPTWDEWPEFARETFQAFRTTDVGRKLII
                     DQNVFIEGTLPMGVVRPLTEVEMDHYREPFLNPVDREPLWRFPNELPIAGEPANIVALV
                     EEYMDWLHQSPVPKLLFWGTPGVLIPPAEAARLAKSLPNCKAVDIGPGLNLLQEDNPDL
                     IGSEIARWLSTLEISG"
     protein_bind    complement(2235..2259)
                     /gene="mutant version of attB"
                     /label=attB2
                     /bound_moiety="BP Clonase(TM)"
                     /note="recombination site for the Gateway(R) BP reaction"
     misc_feature    2269..2857
                     /label=WPRE
                     /note="woodchuck hepatitis virus posttranscriptional 
                     regulatory element"
     primer_bind     complement(2322..2342)
                     /label=WPRE-R
                     /note="WPRE, reverse primer"
     CDS             complement(2740..2751)
                     /codon_start=1
                     /product="Factor Xa recognition and cleavage site"
                     /label=Factor Xa site
                     /translation="IEGR"
     primer_bind     complement(2887..2904)
                     /label=BGH-rev
                     /note="Bovine growth hormone terminator, reverse primer. 
                     Also called BGH reverse"
     polyA_signal    2893..3100
                     /label=bGH poly(A) signal
                     /note="bovine growth hormone polyadenylation signal"
     repeat_region   3108..3248
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
     repeat_region   3108..3237
                     /label=AAV2 ITR
     rep_origin      3323..3778
                     /direction=RIGHT
                     /label=f1 ori
                     /note="f1 bacteriophage origin of replication; arrow 
                     indicates direction of (+) strand synthesis"
     primer_bind     complement(3410..3429)
                     /label=F1ori-R
                     /note="F1 origin, reverse primer"
     primer_bind     3620..3641
                     /label=F1ori-F
                     /note="F1 origin, forward primer"
     primer_bind     complement(3795..3814)
                     /label=pRS-marker
                     /note="pRS vectors, use to sequence yeast selectable 
                     marker"
     primer_bind     3914..3936
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(3974..3992)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        4060..4164
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(4165..4233,4234..5025)
                     /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     complement(4383..4402)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      5196..5784
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     5685..5704
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     repeat_region   join(5846..5856,1..130)
                     /label=AAV2 ITR
                     /note="inverted terminal repeat of adeno-associated virus 
                     serotype 2"
ORIGIN
        1 ctgcgcgctc gctcgctcac tgaggccgcc cgggcaaagc ccgggcgtcg ggcgaccttt
       61 ggtcgcccgg cctcagtgag cgagcgagcg cgcagagagg gagtggccaa ctccatcact
      121 aggggttcct tctagacaac tttgtataga aaagttgtag ttattaatag taatcaatta
      181 cggggtcatt agttcatagc ccatatatgg agttccgcgt tacataactt acggtaaatg
      241 gcccgcctgg ctgaccgccc aacgaccccc gcccattgac gtcaataatg acgtatgttc
      301 ccatagtaac gccaataggg actttccatt gacgtcaatg ggtggagtat ttacggtaaa
      361 ctgcccactt ggcagtacat caagtgtatc atatgccaag tacgccccct attgacgtca
      421 atgacggtaa atggcccgcc tggcattatg cccagtacat gaccttatgg gactttccta
      481 cttggcagta catctacgta ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
      541 acatcaatgg gcgtggatag cggtttgact cacggggatt tccaagtctc caccccattg
      601 acgtcaatgg gagtttgttt tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca
      661 actccgcccc attgacgcaa atgggcggta ggcgtgtacg gtgggaggtc tatataagca
      721 gagctggttt agtgaaccgt cagatccaag tttgtacaaa aaagcaggct gccaccatgg
      781 tcttcacact cgaagatttc gttggggact ggcgacagac agccggctac aacctggacc
      841 aagtccttga acagggaggt gtgtccagtt tgtttcagaa tctcggggtg tccgtaactc
      901 cgatccaaag gattgtcctg agcggtgaaa atgggctgaa gatcgacatc catgtcatca
      961 tcccgtatga aggtctgagc ggcgaccaaa tgggccagat cgaaaaaatt tttaaggtgg
     1021 tgtaccctgt ggatgatcat cactttaagg tgatcctgca ctatggcaca ctggtaatcg
     1081 acggggttac gccgaacatg atcgactatt tcggacggcc gtatgaaggc atcgccgtgt
     1141 tcgacggcaa aaagatcact gtaacaggga ccctgtggaa cggcaacaaa attatcgacg
     1201 agcgcctgat caaccccgac ggctccctgc tgttccgagt aaccatcaac ggagtgaccg
     1261 gctggcggct gtgcgaacgc attctggcgt ctggtggcgg aggctcgggc ggaggtgggt
     1321 cgggtggcgg cggatcaatg gcagaaatcg gtactggctt tccattcgac ccccattatg
     1381 tggaagtcct gggcgagcgc atgcactacg tcgatgttgg tccgcgcgat ggcacccctg
     1441 tgctgttcct gcacggtaac ccgacctcct cctacgtgtg gcgcaacatc atcccgcatg
     1501 ttgcaccgac ccatcgctgc attgctccag acctgatcgg tatgggcaaa tccgacaaac
     1561 cagacctggg ttatttcttc gacgaccacg tccgcttcat ggatgccttc atcgaagccc
     1621 tgggtctgga agaggtcgtc ctggtcattc acgactgggg ctccgctctg ggtttccact
     1681 gggccaagcg caatccagag cgcgtcaaag gtattgcatt tatggagttc atccgcccta
     1741 tcccgacctg ggacgaatgg ccagaatttg cccgcgagac cttccaggcc ttccgcacca
     1801 ccgacgtcgg ccgcaagctg atcatcgatc agaacgtttt tatcgagggt acgctgccga
     1861 tgggtgtcgt ccgcccgctg actgaagtcg agatggacca ttaccgcgag ccgttcctga
     1921 atcctgttga ccgcgagcca ctgtggcgct tcccaaacga gctgccaatc gccggtgagc
     1981 cagcgaacat cgtcgcgctg gtcgaagaat acatggactg gctgcaccag tcccctgtcc
     2041 cgaagctgct gttctggggc accccaggcg ttctgatccc accggccgaa gccgctcgcc
     2101 tggccaaaag cctgcctaac tgcaaggctg tggacatcgg cccgggtctg aatctgctgc
     2161 aagaagacaa cccggacctg atcggcagcg agatcgcgcg ctggctgtcg acgctggaga
     2221 tttccggcta gtaaacccag ctttcttgta caaagtggga attccgataa tcaacctctg
     2281 gattacaaaa tttgtgaaag attgactggt attcttaact atgttgctcc ttttacgcta
     2341 tgtggatacg ctgctttaat gcctttgtat catgctattg cttcccgtat ggctttcatt
     2401 ttctcctcct tgtataaatc ctggttgctg tctctttatg aggagttgtg gcccgttgtc
     2461 aggcaacgtg gcgtggtgtg cactgtgttt gctgacgcaa cccccactgg ttggggcatt
     2521 gccaccacct gtcagctcct ttccgggact ttcgctttcc ccctccctat tgccacggcg
     2581 gaactcatcg ccgcctgcct tgcccgctgc tggacagggg ctcggctgtt gggcactgac
     2641 aattccgtgg tgttgtcggg gaagctgacg tcctttccat ggctgctcgc ctgtgttgcc
     2701 acctggattc tgcgcgggac gtccttctgc tacgtccctt cggccctcaa tccagcggac
     2761 cttccttccc gcggcctgct gccggctctg cggcctcttc cgcgtcttcg ccttcgccct
     2821 cagacgagtc ggatctccct ttgggccgcc tccccgcatc gggaattcct agagctcgct
     2881 gatcagcctc gactgtgcct tctagttgcc agccatctgt tgtttgcccc tcccccgtgc
     2941 cttccttgac cctggaaggt gccactccca ctgtcctttc ctaataaaat gaggaaattg
     3001 catcgcattg tctgagtagg tgtcattcta ttctgggggg tggggtgggg caggacagca
     3061 agggggagga ttgggaagag aatagcaggc atgctgggga gggccgcagg aacccctagt
     3121 gatggagttg gccactccct ctctgcgcgc tcgctcgctc actgaggccg ggcgaccaaa
     3181 ggtcgcccga cgcccgggct ttgcccgggc ggcctcagtg agcgagcgag cgcgcagctg
     3241 cctgcagggg cgcctgatgc ggtattttct ccttacgcat ctgtgcggta tttcacaccg
     3301 catacgtcaa agcaaccata gtacgcgccc tgtagcggcg cattaagcgc ggcgggtgtg
     3361 gtggttacgc gcagcgtgac cgctacactt gccagcgcct tagcgcccgc tcctttcgct
     3421 ttcttccctt cctttctcgc cacgttcgcc ggctttcccc gtcaagctct aaatcggggg
     3481 ctccctttag ggttccgatt tagtgcttta cggcacctcg accccaaaaa acttgatttg
     3541 ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg tttttcgccc tttgacgttg
     3601 gagtccacgt tctttaatag tggactcttg ttccaaactg gaacaacact caactctatc
     3661 tcgggctatt cttttgattt ataagggatt ttgccgattt cggtctattg gttaaaaaat
     3721 gagctgattt aacaaaaatt taacgcgaat tttaacaaaa tattaacgtt tacaatttta
     3781 tggtgcactc tcagtacaat ctgctctgat gccgcatagt taagccagcc ccgacacccg
     3841 ccaacacccg ctgacgcgcc ctgacgggct tgtctgctcc cggcatccgc ttacagacaa
     3901 gctgtgaccg tctccgggag ctgcatgtgt cagaggtttt caccgtcatc accgaaacgc
     3961 gcgagacgaa agggcctcgt gatacgccta tttttatagg ttaatgtcat gataataatg
     4021 gtttcttaga cgtcaggtgg cacttttcgg ggaaatgtgc gcggaacccc tatttgttta
     4081 tttttctaaa tacattcaaa tatgtatccg ctcatgagac aataaccctg ataaatgctt
     4141 caataatatt gaaaaaggaa gagtatgagt attcaacatt tccgtgtcgc ccttattccc
     4201 ttttttgcgg cattttgcct tcctgttttt gctcacccag aaacgctggt gaaagtaaaa
     4261 gatgctgaag atcagttggg tgcacgagtg ggttacatcg aactggatct caacagcggt
     4321 aagatccttg agagttttcg ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt
     4381 ctgctatgtg gcgcggtatt atcccgtatt gacgccgggc aagagcaact cggtcgccgc
     4441 atacactatt ctcagaatga cttggttgag tactcaccag tcacagaaaa gcatcttacg
     4501 gatggcatga cagtaagaga attatgcagt gctgccataa ccatgagtga taacactgcg
     4561 gccaacttac ttctgacaac gatcggagga ccgaaggagc taaccgcttt tttgcacaac
     4621 atgggggatc atgtaactcg ccttgatcgt tgggaaccgg agctgaatga agccatacca
     4681 aacgacgagc gtgacaccac gatgcctgta gcaatggcaa caacgttgcg caaactatta
     4741 actggcgaac tacttactct agcttcccgg caacaattaa tagactggat ggaggcggat
     4801 aaagttgcag gaccacttct gcgctcggcc cttccggctg gctggtttat tgctgataaa
     4861 tctggagccg gtgagcgtgg aagccgcggt atcattgcag cactggggcc agatggtaag
     4921 ccctcccgta tcgtagttat ctacacgacg gggagtcagg caactatgga tgaacgaaat
     4981 agacagatcg ctgagatagg tgcctcactg attaagcatt ggtaactgtc agaccaagtt
     5041 tactcatata tactttagat tgatttaaaa cttcattttt aatttaaaag gatctaggtg
     5101 aagatccttt ttgataatct catgaccaaa atcccttaac gtgagttttc gttccactga
     5161 gcgtcagacc ccgtagaaaa gatcaaagga tcttcttgag atcctttttt tctgcgcgta
     5221 atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa
     5281 gagctaccaa ctctttttcc gaaggtaact ggcttcagca gagcgcagat accaaatact
     5341 gttcttctag tgtagccgta gttaggccac cacttcaaga actctgtagc accgcctaca
     5401 tacctcgctc tgctaatcct gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt
     5461 accgggttgg actcaagacg atagttaccg gataaggcgc agcggtcggg ctgaacgggg
     5521 ggttcgtgca cacagcccag cttggagcga acgacctaca ccgaactgag atacctacag
     5581 cgtgagctat gagaaagcgc cacgcttccc gaagggagaa aggcggacag gtatccggta
     5641 agcggcaggg tcggaacagg agagcgcacg agggagcttc cagggggaaa cgcctggtat
     5701 ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc gtcgattttt gtgatgctcg
     5761 tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc
     5821 ttttgctggc cttttgctca catgtcctgc aggcag
//
