LOCUS       pMAL-pHluorin4          6378 bp    DNA     linear       03-OCT-2025
FEATURES             Location/Qualifiers
     source          1..2688
                     /organism="Cloning vector pMAL-c5X"
                     /mol_type="other DNA"
     gene            15..1163
                     /gene="lacIq"
     -35_signal      15..19
                     /gene="lacIq"
                     /note="lacIq promoter (clockwise); TGGTG"
     -10_signal      40..46
                     /gene="lacIq"
                     /note="lacIq promoter (clockwise); CATGATA"
     CDS             81..1163
                     /gene="lacIq"
                     /codon_start=1
                     /transl_table=11
                     /product="lac repressor (LacI)"
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAE
                     LNYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERS
                     GVEACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSII
                     FSHEDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREG
                     DWSAMSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDT
                     EDSSCYIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNT
                     QTASPRALADSLMQLARQVSRLESGQ"
     -35_signal      1406..1411
                     /gene="malE expression cassette"
                     /note="Ptac promoter (clockwise); TTGACA"
     gene            1406..2688
                     /gene="malE expression cassette"
     -10_signal      1428..1433
                     /gene="malE expression cassette"
                     /note="Ptac promoter (clockwise); TATAAT"
     CDS             1528..3408
                     /gene="malE expression cassette"
                     /note="malE sequence 1528..2628; spacer and factor Xa
                     site 2629..2687; multiple cloning site region 2688..2754"
                     /codon_start=1
                     /product="maltose binding protein (MBP) fusion"
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKL
                     EEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNG
                     KLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWP
                     LIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAF
                     NKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKEL
                     AKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNI
                     PQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISHMS
                     MGGRDIVDGSEFPAGN"
     primer_bind     2587..2610
                     /note="malE primer sequence"
     CDS             2689..3408
                     /dnas_title="pHluorin4"
                     /codon_start=1
                     /product="pHluorin4"
                     /label=pHluorin4
                     /note="mammalian codon-optimized"
                     /translation="MVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLT
                     LKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFK
                     DDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKN
                     GIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEKRDH
                     MVLLEFVTAAGITHGMDELYK"
     misc_feature    3409..3448
                     /gene="malE expression cassette"
                     /note="multiple cloning site (NdeI-SbfI)"
     source          3409..6378
                     /dnas_title="pMAL-pHluorin4"
                     /organism="Cloning vector pMAL-c5X"
                     /mol_type="other DNA"
     gene            3409..3669
                     /gene="malE expression cassette"
     CDS             3409..3455
                     /gene="malE expression cassette"
                     /note="malE sequence 1528..2628; spacer and factor Xa
                     site 2629..2687; multiple cloning site region 2688..2754"
                     /codon_start=1
                     /product="maltose binding protein (MBP) fusion"
                     /translation="MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKL
                     EEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNG
                     KLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWP
                     LIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAF
                     NKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKEL
                     AKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVKDPRIAATMENAQKGEIMPNI
                     PQMSAFWYAVRTAVINAASGRQTVDEALKDAQTNSSSNNNNNNNNNNLGIEGRISHMS
                     MGGRDIVDGSEFPAGN"
     terminator      3467..3510
                     /gene="malE expression cassette"
                     /note="rrnB T1 transcription terminator"
     primer_bind     complement(3524..3547)
                     /note="MCS reverse primer sequence"
     terminator      3642..3669
                     /gene="malE expression cassette"
                     /note="rrnB T2 transcription terminator"
     gene            3718..4648
                     /gene="bla"
     -35_signal      3718..3723
                     /gene="bla"
                     /note="bla promoter (clockwise); TTCAAA"
     -10_signal      3741..3746
                     /gene="bla"
                     /note="bla promoter (clockwise); GACAAT"
     CDS             3788..4648
                     /gene="bla"
                     /note="ampR (confers resistance to ampicillin)"
                     /codon_start=1
                     /product="beta-lactamase"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGY
                     IELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVE
                     YSPVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRL
                     DRWEPELNEAIPNDERDTTMPVAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPL
                     LRSALPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIA
                     EIGASLIKHW"
     rep_origin      4736..5324
                     /note="pMB1 origin of replication (clockwise) (RNAII -35
                     to RNA/DNA switch point)"
     gene            complement(5694..5885)
                     /gene="rop"
     CDS             complement(5694..5885)
                     /gene="rop"
                     /codon_start=1
                     /transl_table=11
                     /product="ROP protein"
                     /translation="MTKQEKTALNMARFIRSQTLTLLEKLNELDADEQADICESLHDH
                     ADELYRSCLARFGDDGENL"
ORIGIN      
        1 ccgacaccat cgaatggtgc aaaacctttc gcggtatggc atgatagcgc ccggaagaga
       61 gtcaattcag ggtggtgaat gtgaaaccag taacgttata cgatgtcgca gagtatgccg
      121 gtgtctctta tcagaccgtt tcccgcgtgg tgaaccaggc cagccacgtt tctgcgaaaa
      181 cgcgggaaaa agtggaagcg gcgatggcgg agctgaatta cattcccaac cgcgtggcac
      241 aacaactggc gggcaaacag tcgttgctga ttggcgttgc cacctccagt ctggccctgc
      301 acgcgccgtc gcaaattgtc gcggcgatta aatctcgcgc cgatcaactg ggtgccagcg
      361 tggtggtgtc gatggtagaa cgaagcggcg tcgaagcctg taaagcggcg gtgcacaatc
      421 ttctcgcgca acgcgtcagt gggctgatca ttaactatcc gctggatgac caggatgcca
      481 ttgctgtgga agctgcctgc actaatgttc cggcgttatt tcttgatgtc tctgaccaga
      541 cacccatcaa cagtattatt ttctcccatg aagacggtac gcgactgggc gtggagcatc
      601 tggtcgcatt gggtcaccag caaatcgcgc tgttagcggg cccattaagt tctgtctcgg
      661 cgcgtctgcg tctggctggc tggcataaat atctcactcg caatcaaatt cagccgatag
      721 cggaacggga aggcgactgg agtgccatgt ccggttttca acaaaccatg caaatgctga
      781 atgagggcat cgttcccact gcgatgctgg ttgccaacga tcagatggcg ctgggcgcaa
      841 tgcgcgccat taccgagtcc gggctgcgcg ttggtgcgga tatttcggta gtgggatacg
      901 acgataccga agacagctca tgttatatcc cgccgttaac caccatcaaa caggattttc
      961 gcctgctggg gcaaaccagc gtggaccgct tgctgcaact ctctcagggc caggcggtga
     1021 agggcaatca gctgttgccc gtctcactgg tgaaaagaaa aaccaccctg gcgcccaata
     1081 cgcaaaccgc ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt
     1141 cccgactgga aagcgggcag tgagcgcaac gcaattaatg taagttagct cactcattag
     1201 gcacaattct catgtttgac agcttatcat cgactgcacg gtgcaccaat gcttctggcg
     1261 tcaggcagcc atcggaagct gtggtatggc tgtgcaggtc gtaaatcact gcataattcg
     1321 tgtcgctcaa ggcgcactcc cgttctggat aatgtttttt gcgccgacat cataacggtt
     1381 ctggcaaata ttctgaaatg agctgttgac aattaatcat cggctcgtat aatgtgtgga
     1441 attgtgagcg gataacaatt tcacacagga aacagccagt ccgtttaggt gttttcacga
     1501 gcaattgacc aacaaggacc atagattatg aaaatcgaag aaggtaaact ggtaatctgg
     1561 attaacggcg ataaaggcta taacggtctc gctgaagtcg gtaagaaatt cgagaaagat
     1621 accggaatta aagtcaccgt tgagcatccg gataaactgg aagagaaatt cccacaggtt
     1681 gcggcaactg gcgatggccc tgacattatc ttctgggcac acgaccgctt tggtggctac
     1741 gctcaatctg gcctgttggc tgaaatcacc ccggacaaag cgttccagga caagctgtat
     1801 ccgtttacct gggatgccgt acgttacaac ggcaagctga ttgcttaccc gatcgctgtt
     1861 gaagcgttat cgctgattta taacaaagat ctgctgccga acccgccaaa aacctgggaa
     1921 gagatcccgg cgctggataa agaactgaaa gcgaaaggta agagcgcgct gatgttcaac
     1981 ctgcaagaac cgtacttcac ctggccgctg attgctgctg acgggggtta tgcgttcaag
     2041 tatgaaaacg gcaagtacga cattaaagac gtgggcgtgg ataacgctgg cgcgaaagcg
     2101 ggtctgacct tcctggttga cctgattaaa aacaaacaca tgaatgcaga caccgattac
     2161 tccatcgcag aagctgcctt taataaaggc gaaacagcga tgaccatcaa cggcccgtgg
     2221 gcatggtcca acatcgacac cagcaaagtg aattatggtg taacggtact gccgaccttc
     2281 aagggtcaac catccaaacc gttcgttggc gtgctgagcg caggtattaa cgccgccagt
     2341 ccgaacaaag agctggcaaa agagttcctc gaaaactatc tgctgactga tgaaggtctg
     2401 gaagcggtta ataaagacaa accgctgggt gccgtagcgc tgaagtctta cgaggaagag
     2461 ttggtgaaag atccgcgtat tgccgccact atggaaaacg cccagaaagg tgaaatcatg
     2521 ccgaacatcc cgcagatgtc cgctttctgg tatgccgtgc gtactgcggt gatcaacgcc
     2581 gccagcggtc gtcagactgt cgatgaagcc ctgaaagacg cgcagactaa ttcgagctcg
     2641 aacaacaaca acaataacaa taacaacaac ctcgggatcg agggaaggat ggtgagcaag
     2701 ggcgaggagc tgttcaccgg ggtggtgccc atcctggtcg agctggacgg cgacgtaaac
     2761 ggccacaagt tcagcgtgcg cggcgagggc gagggcgatg ccaccaacgg caagctgacc
     2821 ctgaagttca tctgcaccac cggcaagctg cccgtgccct ggcccaccct cgtgaccacc
     2881 ctgAGCtacg gcgtgcagtg cttcagccgc taccccgacc acatgaagcg ccacgacttc
     2941 ttcaagtccg ccatgcccga aggctacgtc caggagcgca ccatcagctt caaggacgac
     3001 ggcacctaca agacccgcgc cgaggtgaag ttcgagggcg acaccctggt gaaccgcatc
     3061 gagctgaagg gcatcgactt caagGACgac ggcaacatcc tggggcacaa gctggagtac
     3121 aacttcaacG AGcacCTGgt ctatatcacc gccgacaagc agaagaacgg catcaaggcc
     3181 ATCttcCAGG TGCACcacaa cgtggaggac ggcagcgtgc agctcgccga ccactaccag
     3241 cagaacaccc ccatcggcga cggccccgtg ctgctgcccg acaaccacta cctgCACacc
     3301 cagtccgtgc tgagcaaaga ccccaacgag aagcgcgatc acatggtcTT Cctggagttc
     3361 gtgaccgccg ccgggatcac tcacggcatg gacgagctgt acaagtaagc ggccgcgata
     3421 tcgtcgacgg atccgaattc cctgcaggta attaaataag cttcaaataa aacgaaaggc
     3481 tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg ctctcctgag
     3541 taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg gagggtggcg
     3601 ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca tcctgacgga
     3661 tggccttttt gcgtttctac aaactctttc ggtccgttgt ttatttttct aaatacattc
     3721 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag
     3781 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg
     3841 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt
     3901 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt
     3961 tcgccccgaa gaacgtttcc caatgatgag cacttttaaa gttctgctat gtggcgcggt
     4021 attatcccgt gttgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa
     4081 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag
     4141 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac
     4201 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac
     4261 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac
     4321 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac
     4381 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact
     4441 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg
     4501 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt
     4561 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat
     4621 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta
     4681 gattgatttc cttaggactg agcgtcaacc ccgtagaaaa gatcaaagga tcttcttgag
     4741 atcctttttt tctgcgcgta atctgctgct tgcaaacaaa aaaaccaccg ctaccagcgg
     4801 tggtttgttt gccggatcaa gagctaccaa ctctttttcc gaaggtaact ggcttcagca
     4861 gagcgcagat accaaatact gtccttctag tgtagccgta gttaggccac cacttcaaga
     4921 actctgtagc accgcctaca tacctcgctc tgctaatcct gttaccagtg gctgctgcca
     4981 gtggcgataa gtcgtgtctt accgggttgg actcaagacg atagttaccg gataaggcgc
     5041 agcggtcggg ctgaacgggg ggttcgtgca cacagcccag cttggagcga acgacctaca
     5101 ccgaactgag atacctacag cgtgagctat gagaaagcgc cacgcttccc gaagggagaa
     5161 aggcggacag gtatccggta agcggcaggg tcggaacagg agagcgcacg agggagcttc
     5221 cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt tcgccacctc tgacttgagc
     5281 gtcgattttt gtgatgctcg tcaggggggc ggagcctatg gaaaaacgcc agcaacgcgg
     5341 cctttttacg gttcctggcc ttttgctggc cttttgctca catgttcttt cctgcgttat
     5401 cccctgattc tgtggataac cgtattaccg cctttgagtg agctgatacc gctcgccgca
     5461 gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc ggaagagcgc ctgatgcggt
     5521 attttctcct tacgcatctg tgcggtattt cacaccgcat ataaggtgca ctgtgactgg
     5581 gtcatggctg cgccccgaca cccgccaaca cccgctgacg cgccctgacg ggcttgtctg
     5641 ctcccggcat ccgcttacag acaagctgtg accgtctccg ggagctgcat gtgtcagagg
     5701 ttttcaccgt catcaccgaa acgcgcgagg cagctgcggt aaagctcatc agcgtggtcg
     5761 tgcagcgatt cacagatgtc tgcctgttca tccgcgtcca gctcgttgag tttctccaga
     5821 agcgttaatg tctggcttct gataaagcgg gccatgttaa gggcggtttt ttcctgtttg
     5881 gtcactgatg cctccgtgta agggggattt ctgttcatgg gggtaatgat accgatgaaa
     5941 cgagagagga tgctcacgat acgggttact gatgatgaac atgcccggtt actggaacgt
     6001 tgtgagggta aacaactggc ggtatggatg cggcgggacc agagaaaaat cactcagggt
     6061 caatgccagc gcttcgttaa tacagatgta ggtgttccac agggtagcca gcagcatcct
     6121 gcgatgcaga tccggaacat aatggtgcag ggcgctgact tccgcgtttc cagactttac
     6181 gaaacacgga aaccgaagac cattcatgtt gttgctcagg tcgcagacgt tttgcagcag
     6241 cagtcgcttc acgttcgctc gcgtatcggt gattcattct gctaaccagt aaggcaaccc
     6301 cgccagccta gccgggtcct caacgacagg agcacgatca tgcgcacccg tggccaggac
     6361 ccaacgctgc ccgaaatt
//

